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Survivin is a small protein member of the inhibitor of apoptosis protein family. Its expression occurs in G2/M phase of cell cycle, acting inhibiting apoptosis blocking caspases cascade directly or indirectly, and also controlling cell division. Survivin was found to be overexpressed in breast cancer, and its expression have been associated mostly with poor outcome. The aim of this case-control study was to evaluate the prognostic value of Survivin immunoexpression in early stage (pT1pN0 or pT2pN0) breast cancer. The study was conducted collecting data from 170 women with invasive breast carcinoma. The case group (n=57) and control group (n=113) consisted of patients with and without tumor recurrence, respectively. Immunohistochemical Tissue Microarray analyses were conducted to detect Survivin expression. In addition, molecular classification was done based on immunohistochemistry, and classic clinical and histological breast cancer variables were collected. The findings were submitted to the chi-square test and Fisher’s exact test, followed by multivariate analysis with multinomial logistic regression. The level of statistical significance was set at 5% (p Multinomial logistic regression model. In the first model, variables with p Table 1 - Clinical-pathological characterization of the 170 cases of early stage breast cancer patients with and without recurrence.RecurrenceTotalNoYespAge≤ 55 years8658280,78650,6%51,3%49,1%> 55 years84552949,4%48,7%50,9%Histological subtypeDuctal158104540,24492,9%92,0%94,7%Lobular5502,9%4,4%,0%Others7434,1%3,5%5,3%StagespT1a7520,9024,1%4,4%3,5%pT1b2113812,4%11,5%14,0%pT1c65452038,2%39,8%35,1%pT277502745,3%44,2%47,4%Histological gradeI332850,00519,4%24,8%8,8%II75492644,1%43,4%45,6%III41192224,1%16,8%38,6%Ignored2117412,4%15,1%7,0%Breast surgeryMastectomy8557281,00050,0%50,4%49,1%Conservative85562950,0%49,6%50,9%Sentinel lymph node biopsyNo3220120,35318,82%17,7%18,2%Yes137934481,5%82,3%80,0%Ignored1010,6%0,0%1,8%ChemotherapyNo5135160,69730,0%31,0%28,1%Yes119784170,0%69,0%71,9%TrastuzumabNo156105510,76192,3%92,9%91,1%Yes13857,7%7,1%8,9%Hormone therapyNo2918110,58117,1%15,9%19,3%Yes141954682,9%84,1%80,7% Table 2 - Patterns of Survivin expression in tumors of patients with breast cancer with and without recurrence.RecurrenceTotal**NoYespSubcellular localizationAbsent4220,9422,7%2,1%3,8%Cytoplasmic89573260,1%60,0%60,4%Nuclear2315815,5%15,8%15,1%Cytoplasmic and Nuclear32211121,6%22,1%20,8%IntensityAbsent4220,0212,7%2,1%3,8%Weak7353*2049,3%55,8%37,7%Moderate62382441,9%40,0%45,3%Strong927*6,1%2,1%13,2%Percentage of expression≤ 50%10977320,00673,6%81,1%60,4%> 50%39182126,4%18,9%39,6%*p Table 3 - Cox survival regression model of patients diagnosed and treated for invasive breast carcinoma.p-ValorAdjusted ORModel 1Histological grade*0,0072,108Survivin - intensity*0,0231,850Survivin - percentage of expression0,1001,876Breast cancer subtype classified by immunohistochemistry0,2631,126Model 2Survivin - percentage of expression*0,0162,290Breast cancer subtype classified by immunohistochemistry0,0931,170Model 3Breast cancer subtype classified by immunohistochemistry0,2411,107 Citation Format: Heloisa OM Belchior, Victor P Andrade, Marcos VA Lima, Maria do PSS Cunha, Monique BC Lemos, Paulo GB Silva, Isabelle JL Silva-Fernandes. Survivin immunoexpression: An independent prognostic marker of recurrence in early-stage breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-61.

Background. We recently reported that the biosynthetic mevalonate (MVA) pathway that produces cholesterol and isoprenoid intermediates, regulates the YAP/TAZ (Y/T) transcriptional co-activators to promote resistance to treatment regimens that effectively inhibit HER2 signaling in HER2+ BC. We further showed that the mTORC1 complex and survivin protein, which contribute to HER2-elicited oncogenic signaling in HER2-driven BC cells, are alternatively activated by the MVA pathway-Y/T axis in anti-HER2 therapy resistant cell models. Of note, other recent reports also showed that increased Y/T activity enables resistant cells to proliferate when other oncogenic pathways (e.g., RAS, EGFR, and FGFR) were effectively inhibited. Here, we sought to further determine the molecular underpinnings of MVA-Y/T axis-driven anti-HER2 therapy resistance to discover novel therapeutic targets and predictive biomarkers for HER2+ BC.Methods. SKBR3 HER2+ BC parental (P) cells and their lapatinib plus trastuzumab (LT) resistant (LTR) derivatives with sustained HER2 inhibition were treated with the MVA pathway inhibitor simvastatin (Sim), with or without the MVA metabolite to rescue Sim’s inhibitory effects. P and LTR cells were also transfected with control or combined Y/T siRNAs. The transcriptomes of all treatment groups were assessed by RNA-seq. Integrative bioinformatics analyses were used to identify differentially expressed (DE) genes and gene sets with functional annotations in LTR vs. P cells upon different interventions.Results. We found that cell cycle and cell proliferation processes were among the top common DE molecular signatures preferentially downregulated (DN) in LTR vs. P cells by both Sim and Y/T knockdown (KD). The top common genes preferentially DN in LTR vs. P cells include the cell cycle regulatory genes CDCA3 and ERCC6L, and the nucleotide metabolism genes TYMS and RRM2. Interestingly, 20% of the genes preferentially DN in LTR vs. P cells by Sim or Y/T KD were predicted to be direct Y/T transcriptional targets based on previously reported ChIP-seq data (PMID: 26258633). Of the Y/T-dependent genes, a significant enrichment of Sim-repressed genes was observed in both P and LTR cells (P = 1.2e-115 and P = 5.5e-138, respectively). The proportion of these enriched genes was higher in LTR vs. P cells (61% vs. 29%). Of note, we found that the global inhibited genes in LTR cells upon Sim or Y/T KD were significantly enriched for the genes DN by short-term LT treatment in P cells (P < 2.2e-16). Likewise, of the genes nominated as putative molecular players in the MVA pathway-Y/T-mediated resistance, BIRC5 (survivin), CDC6, KIF2C, RRM2, and TYMS were recently also reported to be DN in HER2+ tumors treated with neo-adjuvant LT in the PAMELA trial (NCT01973660), a finding that is in line with what we observed in our P cells treated with short-term LT. Conclusions. Upon acquisition of resistance to sustained HER2 inhibition, the MVA pathway-Y/T axis takes over the regulation of pro-proliferative transcriptional programs that are generally downstream of HER2 signaling in treatment-naïve HER2+ BC. The MVA pathway-Y/T axis leads to Y/T-driven transcriptional reprogramming, an emerging mechanism of therapy resistance to anti-HER2 and other targeted therapies that warrants further investigation. The identification of multiple cell cycle related processes as putative targets of the MVA pathway-Y/T axis presents additional targetable vulnerabilities and implies that inhibitors of Y/T and cell cycle checkpoints may help circumvent anti-HER2 resistance in the clinical setting. Citation Format: Vidyalakshmi Sethunath, Xiaoyong Fu, Pamela L Luna, Sarmistha Nanda, Martin Shea, Jamunarani Veeraraghavan, Carmine De Angelis, Huizhong Hu, Chad Shaw, Mothaffar Rimawi, C. Kent Osborne, Rachel Schiff. Insights into the molecular underpinnings of the mevalonate pathway-YAP/TAZ-driven anti-HER2 therapy resistance in HER2+ breast cancer (BC) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-29.

RNA-binding motif 38 (RBM38) is a member of a protein family with a highly conserved RNA-binding motif and has been shown to regulate mRNA processing, stability, and translation. Survivin is an essential modulator of apoptotic and nonapoptotic cell death as well as a stress responder. Survivin mRNA is the fourth most frequently overexpressed transcript in the human cancer transcriptome, and its aberrant expression is associated with chemo-/radioresistance and poor prognosis. In this study, we examined whether survivin expression is regulated by RBM38. RBM38 bound to survivin 3′-untranslated region and suppressed miRNA let-7b from binding to and degrading survivin mRNA, leading to increased survivin expression. RBM38 interacted with argonaute-2 (AGO2) and facilitated miR-203a–mediated degradation of survivin mRNA, leading to decreased survivin expression. Due to the abundance of let-7b over miR-203a, RBM38 ultimately increased survivin expression in HCT116 and MCF7 cells. In addition, Ser-195 in RBM38 interacted with Glu-73/-76 in AGO2, and Pep8, an eight-amino acid peptide spanning the region of Ser-195 in RBM38, blocked the RBM38–AGO2 interaction and inhibited miR-203a–mediated mRNA degradation, leading to enhanced survivin expression. Furthermore, Pep8 cooperated with YM155, an inhibitor of survivin, to suppress tumor spheroid growth and viability. Pep8 sensitized tumor cells to YM155-induced DNA damage in an RBM38-dependent manner. Together, our data indicate that RBM38 is a dual regulator of survivin and that Pep8/YM155 may be therapeutically explored for tumor suppression. Significance: These findings show that RBM38 exerts opposing effects on survivin expression via two miRNAs, and disruption of the RBM38–AGO2 complex by an eight-amino acid peptide sensitizes tumor spheroids to survivin inhibitor YM155.

Substantial evidence has shown that overexpression of the inhibitor of apoptosis protein (IAP) survivin in human tumors correlates significantly with treatment resistance and poor patient prognosis. Survivin serves as a radiation resistance factor that impacts the DNA damage response by interacting with DNA-dependent protein kinase (DNA-PKcs). However, the complexity, molecular determinants, and functional consequences of this interrelationship remain largely unknown. By applying coimmunoprecipitation and flow cytometry-based Förster resonance energy transfer assays, we demonstrated a direct involvement of the survivin baculovirus IAP repeat domain in the regulation of radiation survival and DNA repair. This survivin-mediated activity required an interaction of residues S20 and W67 with the phosphoinositide 3-kinase (PI3K) domain of DNA-PKcs. In silico molecular docking and dynamics simulation analyses, in vitro kinase assays, and large-scale mass spectrometry suggested a heterotetrameric survivin–DNA-PKcs complex that results in a conformational change within the DNA-PKcs PI3K domain. Overexpression of survivin resulted in enhanced PI3K enzymatic activity and detection of differentially abundant phosphopeptides and proteins implicated in the DNA damage response. The survivin–DNA-PKcs interaction altered the S/T-hydrophobic motif substrate specificity of DNA-PKcs with a predominant usage of S/T-P phosphorylation sites and an increase of DNA-PKcs substrates including Foxo3. These data demonstrate that survivin differentially regulates DNA-PKcs-dependent radiation survival and DNA double-strand break repair via formation of a survivin–DNA-PKcs heterotetrameric complex. Significance: These findings provide insight into survivin-mediated regulation of DNA-PKcs kinase and broaden our knowledge of the impact of survivin in modulating the cellular radiation response. See related commentary by Iliakis, p. 2270

In breast cancer, both incidence and mortality have been increasing. Triple negative breast cancer (TNBC), one of breast cancer subtypes, is a heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor type 2 (HER2). Due to the lack of expression of therapeutic targets, new therapeutic strategy and elucidation the mechanism of action in TNBC are strongly desired. Histone acetyltransferases E1A binding protein (p300) and CREB binding protein (CBP) are coactivators of a large number of essential transcription factors that contribute to cell survival and proliferation. p300/CBP (CREBBP) interacts with transcription factors and other proteins through histone acetyltransferase (HAT) and regulates several gene expression. Dysregulation of CREBBP HAT activity could contribute to various diseases, including cancer. It is reported that the high expression of p300 correlates with poor prognosis in breast cancers. However, the mechanism of CREBBP HAT activity remains unclear. We, therefore, focused on CREBBP and clarified its important mechanism in TNBC. In breast cancer cell lines, p300 expression was higher than the normal mammary epithelial cell (MCF10A) in mRNA and protein levels and positively correlated with that of CBP. To evaluate the roles of p300 and CBP in cell survival and proliferation, the loss of function studies was conducted. In TNBC cell line (MDA-MB-231), cell survival and proliferation significantly reduced in p300 knockdown cells. The reduction in cell survival and proliferation was also observed using p300 inhibitor (L002). To examine the mechanism of action for the reduction of cell survival and proliferation in p300 inhibition, cell cycle assay and apoptosis assay were performed. We found that the cell-cycle progression was arrested at the cell cycle G2/M phase and apoptosis induction in p300 knockdown cells. These results suggest that cell cycle G2/M phase arrest and the late stage of apoptosis are caused by p300 inhibition and p300 inhibition enhances the anti-tumor effect. To identify a mechanism responsible for the reduction of cell survival and proliferation in p300 inhibition, antibody array was performed. As a result of this assay and TCGA patients cohorts analysis, we identified survivin (BIRC5), a member of the inhibitor of apoptosis (IAP) family, as a functional novel biomarker of TNBC with significantly higher expression. These results suggest that suppression of BIRC5 through CREBBP inhibitor result in G2/M phase cell cycle arrest and apoptotic cell death in vitro and in vivo. In conclusion, CREBBP-BIRC5 transcriptional complex could be a novel therapeutic target for TNBC. Citation Format: Sunao Tanaka, Natsuki Wariishi, Nobuko Kawaguchi-Sakita, Souichi Adachi, Masakazu Toi, Yasuhiko Kamikubo. The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-06.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a large protein kinase and a member of the PI3K-related family of protein kinases that also includes ATM and ATR. DNA-PKcs is a unique evolutionary endowment of higher eukaryotes, as it is absent in lower eukaryotes. It is central to the processing of DNA double-strand breaks by classical nonhomologous end-joining, where through interaction with the Ku70/Ku80 heterodimer it generates the DNA-PK holoenzyme. DNA-PK coordinates and regulates the joining of DNA ends through essential structural contributions and by direct phosphorylation of key repair factors, including itself. Recent structural studies advance our understanding of the functions of this giant enzyme and reveal functional complexity and sophistication compatible with a broad spectrum of activities. Along these lines, the observations reported in the article by Güllülü and colleagues in this issue of Cancer Research reveal intriguing new facets in the regulation of DNA-PKcs and open horizons for further exciting research. Güllülü and colleagues found that in addition to known modes of regulation, DNA-PKcs is also regulated by a direct interaction with survivin. The observations expand the functional and regulatory spectrum of this intriguing kinase and suggest contributions to DNA damage response that remain to be characterized. They formed the foundations for the development of drugs disrupting this interaction, thereby potentially sensitizing tumor cells to radiation. See related article by Güllülü et al., p. 2304

Mutations in ARID1A, encoding a subunit of the BAF chromatin remodeling complex, rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward human genetic model systems. Here, CRISPR/Cas9 knockout of ARID1A in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation but impaired Wnt/β-catenin signaling. Genetic Wnt pathway activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative essential pathways of ARID1A-mediated transformation. ARID1A loss induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1 regulation of multiple mitotic regulatory genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to BIRC5/survivin inhibition, functionally implicating BIRC5/survivin as an essential mediator of proliferation following initial ARID1A loss. However, CRISPR inactivation of ARID1A in established gastric cancer cell lines did not elicit sensitivity to BIRC5/survivin inhibitors, suggesting that this pathway may be selectively required during the early establishment of tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, indicating the general utility of organoid-based forward genetic cancer analysis in human cells. Citation Format: Yuan-Hung Lo, Kevin S. Kolahi, Yuhong Du, Chiung-Ying Chang, Andrey Krokhotin, Ajay Nair, Walter D. Sobba, Kasper Karlsson, Sunny J. Jones, Teri A. Longacre, Amanda T. Mah, Alexandra Sockell, Jose A. Seoane, Jin Chen, Jonathan S. Weissman, Christina Curtis, Andrea Califano, Haian Fu, Gerald R. Crabtree, Calvin J. Kuo. A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 123.

Introduction: Multiple myeloma (MM) is a plasma cell disorder characterized by clonal expansion and accumulation of immature undifferentiated neoplastic cells in the bone marrow (BM). Recent advances in fluorochrome chemistries, flow cytometry (FACS) instrumentation and rapid tissue preservation methodologies have created opportunities for broader adoption of FACS in the clinical management of multiple myeloma patients. Methods: We have developed a 10 color, 2 tube FACS assay based on recommendations made by the European Myeloma Network for phenotyping plasma cell gammopathies to identify abnormal blasts in multiple myeloma patient bone marrow aspirates. The assay includes essential plasma cell markers CD138, CD38, CD56 and CD19 to differentiate abnormal blasts from reactive plasma cells. Use of a multi epitope fluorochrome conjugated anti-CD38 antibody enables CD38 detection in patients on anti-CD38 therapy. Results: Quantification of cell surface HLA-A2 and intracellular survivin protein was made possible using fluorochrome conjugated antibodies in combination with bead standards. The SmartTube™ proteomic stabilizer system for rapid preservation was used to freeze BM aspirate specimens for later analysis at locations with appropriate FACS equipment and trained staff. Consistent with previously published literature, patient survivin levels were elevated on abnormal MM blasts in comparison to survivin levels on non-blast immune cells. Conclusion: We have developed a robust FACS methodology that would be easy to implement in the clinical trial and development setting with minimal training. The assay can consistently identify abnormal blasts in BM samples of multiple myeloma patients and quantify intracellular and cell surface biomarkers related to MM disease or disease treatment responses. Citation Format: Tejaswini Hardas, Hyun-Jeong Ra, James Sheridan, Maria Kovalenko, Catherine Tribouley. Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 408.

Breast cancer is the most common cancer diagnosed in women. Endoplasmic reticulum stress (EnR stress) and the related unfolded protein response (UPR) are activated in breast cancer cells and can promote cell survival and endocrine resistance. TMEM33 is a novel transmembrane protein that resides in the endoplasmic reticulum (EnR) and has been shown to activate the PERK and IRE1α branches of the UPR. However, the underlying mechanism of action of this EnR resident protein TMEM33 and the cellular functions that it regulates remain largely unknown. In this study, we show that overexpression of TMEM33 induces robust cell death in breast cancer cells. TMEM33 overexpression strongly activates UPR associated pro-death JNK-p53 signaling. We also observed a significant inhibition of the downstream survivin, which blocks cell death activation by binding to caspases and inhibiting their activation. We further show that the blockage of JNK activation with either an inhibitor or overexpression of survivin, protects cells against TMEM33 induced apoptosis. In addition, we show that TMEM33 overexpression induces autophagy in breast cancer cells. Inhibition of autophagy with using either the inhibitor chloroquine or knockdown of the Atg5 gene, further sensitizes breast cancer cells to the effects of TMEM33 overexpression. Cell death induced by TMEM33 is also decreased by overexpression of the autophagy gene Beclin 1. The findings in this study demonstrate that the novel EnR resident protein TMEM33 induces cell death by activating IRE1α-JNK-p53-survivin signaling in breast cancer cells. Concurrently, autophagy is also activated by TMEM33, and functions as a pro-survival mechanism. Cell fate reflects the balance between the pro-death and pro-survival activities as regulated by TMEM33. Citation Format: Clarke R, Hu R, Zhang X, Hilakivi-Clarke L, Kasid U. Transmembrane protein 33 (TMEM33) induces apoptosis via UPR signaling and autophagy in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-10-02.

Background: Approximately 15% of all breast cancers are categorized as triple negative (TNBC) for which the only chemotherapy is known to be effective, yet often fails to achieve remission. Nuclear exporter XPO1 (Exportin1 or CRM1) is a promising target for cancer therapy that mediates the transport of multiple tumor suppressors and cell cycle regulators that have been known to be relevant predictors in the mechanism and severity of TNBC. Given the pressing need for novel therapies for this disease, we sought to determine the antitumor effects of selinexor, a novel inhibitor of nuclear export, on triple negative breast cancers in vitro and in vivo as well as to address its mechanism of action. Methods: 26 breast cancer cell lines of different breast cancer subtypes were treated with selinexor in vitro. Using cell proliferation assays the half maximal inhibitory concentration (IC50) was calculated using isobologram curves after 3 days of treatment; sensitivity was defined as IC50 Results: Selinexor demonstrated growth inhibition in all fourteen TNBC cell lines tested; TNBC cell lines were more sensitive to selinexor (median IC50 44nM, range 11 - 550nM), compared to ER+ cells lines (median IC50 of 13000 nM, range of 40nM - > 1000 nM; P=0.017). Treatment with selinexor decreased expression levels of XPO1, as well as survivin and XIAP, and induced apoptosis. In multiple TNBC cell lines selinexor was synergistic with paclitaxel, carboplatin, eribulin and doxorubicin in vitro (median combination index 0.6, range 0.5-0.8). Selinexor as a single agent reduced tumor growth in vivo in 4 of 5 different TNBC PDX models with a median tumor growth inhibition ratio score (T/C) of 48% (range 34-59%) and demonstrated greater antitumor efficacy in combination with paclitaxel or eribulin with an average T/C score of 27% and 12% respectively. Conclusions: Selinexor is a promising therapeutic agent for triple negative breast cancer and it has potential as a combination agent with standard chemotherapy. Citation Format: Paez Arango N, Evans KW, Zhao M, Yuca E, Scott SM, Janku F, Ueno NT, Tripathy D, Kim C, Naing A, Funda M-B. Selinexor, a selective inhibitor of nuclear export, demonstrates efficacy in preclinical models of triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-01.

In the original version of [this article][1] ([1][2]), the Western blot image in Fig. 4F was inadvertently taken from the results of a different experiment. The authors provided the Western blot image from the correct experiment and the error has been corrected in the latest online HTML and PDF

Androgen receptor signaling fuels prostate cancer and is a major therapeutic target. However, mechanisms of resistance to therapeutic androgen ablation are not well understood. Here, using a prostate cancer mouse model, Ptenpc−/−, carrying a prostate epithelial-specific Pten deletion, we show that the αvβ6 integrin is required for tumor growth in vivo of castrated as well as of noncastrated mice. We describe a novel signaling pathway that couples the αvβ6 integrin cell surface receptor to androgen receptor via activation of JNK1 and causes increased nuclear localization and activity of androgen receptor. This downstream kinase activation by αvβ6 is specific for JNK1, with no involvement of p38 or ERK kinase. In addition, differential phosphorylation of Akt is not observed under these conditions, nor is cell morphology affected by αvβ6 expression. This pathway, which is specific for αvβ6, because it is not regulated by a different αv-containing integrin, αvβ3, promotes upregulation of survivin, which in turn supports anchorage-independent growth of αvβ6-expressing cells. Consistently, both αvβ6 and survivin are significantly increased in prostatic adenocarcinoma, but are not detected in normal prostatic epithelium. Neither XIAP nor Bcl-2 is affected by αvβ6 expression. In conclusion, we show that αvβ6 expression is required for prostate cancer progression, including castrate-resistant prostate cancer; mechanistically, by promoting activation of JNK1, the αvβ6 integrin causes androgen receptor–increased activity in the absence of androgen and consequent upregulation of survivin. These preclinical results pave the way for further clinical development of αvβ6 antagonists for prostate cancer therapy. Cancer Res; 76(17); 5163–74. ©2016 AACR.

Background: We previously reported that telapristone acetate (TPA), an anti-progestin, significantly suppressed mammary tumor formation and growth accelerated by progesterone (P4) and medroxyprogesterone acetate (MPA) in N-methyl-N-nitrosourea (MNU)-induced carcinogenesis model[1]. This suggests TPA holds promise for the prevention and treatment of human breast cancer. The purpose of this study is to identify the genes modulated by TPA in rat mammary ER/PR+ tumors, which may be translated into a therapeutic biomarker of TPA therapy in ER/PR+ breast cancer. Methods: Rat mammary tumors from MNU carcinogenesis were used for study [1]. Treatment groups were: control, MPA, P4, MPA+TPA, and P4+TPA. Doses were 30mg of TPA and 25mg of P4 or MPA (90 day release pellets). Tumor epithelium was isolated by laser capture microdissection and RNA extracted. 100 ng of RNA per sample was used for targeted -gene expression profiling by nCounter Gene Expression analysis (Nanostring Inc.). 112 genes related to PR, GR, AR signaling were selected by MetaCore pathway analysis (Thomson Reuters, Inc.). The data was normalized by positive control and RNA content using nSolver. The genes significantly modulated by hormones and TPA were reported as means ± standard deviations (SD) for each group as well as a p-value for pairwise t-test comparison. Results: mRNA expression, log mean (log SD).Control vs. P4GeneControlP4pCHD51.5 (0.6)0.7 (0.5)0.03P4 vs. P4+TPAGeneP4P4+TPApSurvivin0.7 (0.5)3.9 (0.2)0.02KI677.6 (0.3)7.1 (0.3)0.02FANCA4.3 (0.2)3.8 (0.3)0.03EZH25.4 (0.2)5.1 (0.2)0.03RFC35.6 (0.2)5.3 (0.2)0.03C.MYC5.9 (0.4)5.5 (0.3)0.04TK15.2 (0.3)4.8 (0.2)0.05Control vs. MPAGeneControlMPApFKBP54.7 (0.4)5.6 (0.6)0.00TNFSF112.0 (0.2)2.8 (0.9)0.02TEK3.7 (0.5)3.1 (0.5)0.03PLZF2.8 (0.9)4.0 (1.1)0.03EPAS15.5 (0.5)4.9 (0.4)0.04MPA vs. MPA+TPAGeneMPAMPA+TPApEGFR6.4 (0.2)6.0 (0.4)0.02SNAI25.0 (0.3)4.6 (0.4)0.04AR4.2 (0.8)5.0 (0.7)0.04Endothelin-15.0 (0.7)3.1 (0.6)0.04 Compared to the control, P4 significantly reduced CHD5 expression, a tumor suppressor in a mouse model, and its down-regulation contributes to the development and progression of human breast cancer. Comparing the P4 and P4+TPA groups, the genes involved in cell proliferation and DNA repair (C-MYC, FANCA, Ki67, RFC3, and TK1), EZH2 (stem cell self-renewal), and anti-apoptosis (survivin) were significantly down-regulated by TPA. On the other hand, MPA induced the up-regulation of FKBP5 (androgen-regulated protein), PLZF (GR responsive gene), TEK (angiogenesis-regulating gene) and TNFSF11 (RANKL) compared to the control. Comparing the MPA and MPA+TPA groups, TPA significantly reduced the expression of EGFR, SNAI2 and endothelin-1 (EMT initiation or maintenance) and increased AR expression. Conclusions: TPA inhibits P4/PR mediated cell proliferation, and increases apoptosis. The tumorigenic effect of MPA is likely through AR and GR rather than a PR specific manner. In addition to the PR, MPA binds with high affinity to AR and GR, and therefore has the potential to elicit actions distinct to those of P4, which does not bind AR. [1] Lee O, et al. Cancer Res 2013 vol. 73 no. 24 Supplement P4-11-02. Citation Format: Lee O, Clare SE, Ivancic D, Scholtens DM, Khan SA. Telapristone acetate suppresses the expression of genes involved in cell proliferation, stem cell self-renewal, and anti-apoptosis in MNU-induced rat mammary tumors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-11-01.

STAT3 offers an attractive target for cancer therapy, but small-molecule inhibitors with appealing pharmacologic properties have been elusive. Here, we report hydroxamic acid–based and benzoic acid–based inhibitors (SH5-07 and SH4-54, respectively) with robust bioactivity. Both inhibitors blocked STAT3 DNA-binding activity in vitro and in human glioma, breast, and prostate cancer cells and in v-Src–transformed murine fibroblasts. STAT3-dependent gene transcription was blocked along with Bcl-2, Bcl-xL, Mcl-1, cyclin D1, c-Myc, and survivin expression. Nuclear magnetic resonance analysis of STAT3-inhibitor complexes defined interactions with the SH2 and DNA-binding domains of STAT3. Ectopic expression of the SH2 domain in cells was sufficient to counter the STAT3-inhibitory effects of SH4-54. Neither compound appreciably affected STAT1 or STAT5 DNA-binding activities, STAT3-independent gene transcription, or activation of a panel of oncogenic kinases in malignant cells. Each compound decreased the proliferation and viability of glioma, breast, and prostate cancer cells and v-Src–transformed murine fibroblasts harboring constitutively active STAT3. Further, in mouse xenograft models of glioma and breast cancer, administration of SH5-07 or SH4-54 effectively inhibited tumor growth. Our results offer preclinical proof of concept for SH5-07 and SH4-54 as candidates for further development as cancer therapeutics. Cancer Res; 76(3); 652–63. ©2015 AACR.

Background: BIRC5/survivin is highly expressed in cancer cells and represents a promising therapeutic target. In order to complete a comprehensive survey of its splice variants, we applied a direct RT-PCR approach as well as next generation sequencing (NGS) to tumor cell cDNAs. We have identified 10 novel splice junctions and three novel splice variants of survivin. Methods: Survivin transcripts were amplified from A1207 (glioblastoma) HeLa (cervical adenocarcinoma) and U87 (glioblastoma) cDNAs. For manual identification of splice forms, individual products were subcloned into TOPO vector and sequenced. For NGS analysis, RT-PCR products used to generate libraries which were subjected to sequencing, after which resulting reads were mapped against the human reference genome and assembled by Trinity. Results: Survivin RT-PCR products from A1207 cDNA were isolated and selected for TOPO cloning/sequencing. Of seventeen clones, the canonical isoform was represented by 4, and known variants including Δex3, 2B and 3B were represented by 5. Two novel sequences were identified: 1) 2B/Δex3, which contains a 68 bp shuffled exon 4/3 segment between exons 3 and 4 plus the 2B insert, and 2) 4B, in which exon 4 is extended by 27 bp at its 5' end. Both of these transcripts are predicted to encode full-length proteins: 2B/Δex3 contains the 23 aa 2B insert plus Δex3's unique C-terminus; 4B contains a novel 9 aa motif embedded in survivin's C-terminal alpha helix. RT-PCR products from A1207, HeLa and U87 templates subjected to NGS yielded five full-length BIRC5 contigs, including the canonical isoform, Δex3, 2B, 3B, and the predicted variant 2B/3B. To identify novel splice junctions, sequences were searched excluding canonical exon boundaries. The resulting 151 reads represented 10 novel exon junctions, among which the greatest proportion consisted of exon 1 and 3 (71 reads, 47%), or an extension of exon 2B by 32 bp at its 3' end (35 reads, 23%). Two variants contained the extended version of exon 4 seen in isoform 4B. A primer annealing within exon 4B's unique 27 bp was subsequently used for RT-PCR to validate the expression of the full-length isoform in HeLa, U87, A1207 and HEK293T cells. Conclusions: We have identified multiple novel splice variants of survivin, including Δex3/2B, 4B, and Δex3/4B. Proteins encoded by these transcripts potentially have pro- or anti-oncogenic functions, and further studies are needed to delineate their role in survivin biologies. Citation Format: Sheila Figel, Meaghan Birkemeier, Jesse Luce, Prashant Singh, Jianmin Wang, Cheryl Frank, Sanam Dharma, Emma Steinmetz, Michael Ciesielski, Robert Fenstermaker. Identification of novel BIRC5/survivin splice variants in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5873.

Objective Yin Yang 1 (YY1) is a transcription factor with a dual role in cancer genesis. Hematological malignancies, including non-Hodgkin lymphomas (NHL), are often characterized by a frequent development of resistance to therapy and a high rate of tumor recurrence. Our previous observations showed that YY1 is overexpressed in hematological tumors. The aim of this study was to investigate the functional role of YY1 in anti-cancer therapy induced cell death and to identify YY1 downstream resistant factors. Experimental Designs The NHL Raji cell line was used as a cellular model. The expression of YY1 was assessed by q-PCR and WB. YY1 knock-down (KD) was achieved using retroviral shRNA. Viability was measured by both the MTT assay and the Trypan Blue live/dead cell count. Cell death was induced following Vincristine and Doxorubicin treatments. The following analyses were used in this study: the JASPAR database, the Encyclopedia of DNA Elements (ENCODE) and the Cancer Genome Atlas (TCGA) datasets relative to NHL patients have been analyzed to assess the expression levels of YY1 and apoptotic genes, according to tumor stages and patients' clinical-pathological features. Results Silencing of YY1 in Raji cells resulted in increased sensitivity to both Vincristine and Doxorubicin treatments and augmented apoptosis. Jaspar analysis identified several potential YY1 transcriptional targets involved in cellular death regulation. As a result of q-PCR screening and validation, Survivin has been identified as a potential YY1 target, as it was significantly downregulated following YY1 KD. Bioinformatic analysis of deposited Chip-Seq databases in ENCODE revealed that YY1 is able to target Survivin transcriptional regulatory regions in different cancer cell lines, as well as in normal B lymphocytes. The TCGA-deposited datasets identified specific clusters of YY1 expression significantly correlated with Survivin. Conclusions Our findings demonstrated that (1) YY1 silencing resulted in the sensitization of Raji tumor cells to drug-induced apoptosis (2) q-PCR-mediated screening of putative YY1 targets led to the identification of Survivin as a potential YY1 target, as when YY1 was KD, Survivin levels were strongly downregulated (3) YY1 might be positively regulating Survivin expression (4) Survivin could be a potential target of YY1 in NHL and (5) YY1 and Survivin in NHL may represent two novel diagnostic and prognostic biomarkers to assess patients' response to chemotherapy. Citation Format: Silvia Vivarelli, Luca Falzone, Saverio Candido, Benjamin Bonavida, Massimo Libra. Oncogenic role of the transcription factor YY1 and its target Survivin in non-Hodgkin's lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4687.

One of the major challenges in colon cancer chemotherapy is multidrug resistance (MDR), which is typically mediated by the overexpression of ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp, ABCB1, MDR1). A number of P-gp inhibitors have been developed, however, none of these compounds have improved chemotherapeutic efficacy due to undesirable pharmacokinetic profiles or adverse effects, resulting in limited clinical success. Therefore, alternative approaches are urgently needed to circumvent MDR cancer. In previous study, a series of synthesized analogs of MX-106, as anti-cancer drugs targeting survivin, exhibited collateral sensitivity (CS) effect to P-gp overexpressing MDR colon cancer cells as well as ABCB1 gene transfected cells, reflected by more than 10-fold cytotoxic effect in P-gp positive MDR cell lines compared to drug sensitive cell lines. Among the analogs, MX-106-4C was identified as the leading compound with the most potent selective toxicity to P-gp overexpressing cells. MX-106-4C-induced CS effect was observed in both intrinsic and acquired P-gp overexpressing colon cancer cells, which was only partially reversed with the presence of a P-gp inhibitor. Nevertheless, this CS effect was abolished in ABCB1-knockout cells, indicating that the selective cytotoxicity was P-gp expression dependent, but only partially related to P-gp function. Furthermore, we found that MX-106-4C did not significantly affect P-gp ATPase activity or drug accumulation and efflux in P-gp-overexpressing cells. In P-gp overexpressing colon cancer cells, short-term (up to 72 h) incubation of MX-106-4C significantly down regulated P-gp expression at transcriptional level but not protein level, whereas long-term (14 d) incubation of MX-106-4C significantly down regulated P-gp protein expression and re-sensitized MDR colon cancer cells to doxorubicin. These findings suggested an indirect interaction and regulation between MX-106-4C and P-gp. Further study revealed that the selective cytotoxic effects of MX-106-4C were associated with cell cycle arrest at G1 phase and apoptosis through the downregulation of CDK4. Overall, this study demonstrates that MX-106-4C selectively kills P-gp positive MDR colon cancer cells and indirectly regulates P-gp, which provides a clue for CS compound design and a novel strategy to obviate P-gp-mediated colon cancer MDR by re-sensitizing heterogeneous tumors with CS agents. Citation Format: Zi-Ning Lei, Zhongzhi Wu, Qiu-Xu Teng, Min Xiao, Wei Li, John N. Wurpel, Zhe-Sheng Chen. Selective toxicity of MX-106-4C, a survivin inhibitor, in P-glycoprotein-mediated multidrug resistant colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5264.

Introduction: Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. Therefore, it is a potential drug target. However, targeting survivin is still challenge and small molecule inhibitors failed in clinical trials. It's very important to understand how survivin contributes to tumor development. In this study, we investigated the role of survivin in ovarian cancer using ovarian cancer cell lines in vitro and mouse models in vivo and examined how survivin contributes to ovarian primary ovarian tumor growth and metastasis by examining epithelial to mesenchymal transition (EMT). Methods: To define the role of survivin in EMT in ovarian cancer cells, we established survivin knockout cell lines using lentiviral CRISPR/Cas9 nickase and pharmacologically inhibited survivin with a novel inhibitor MX106. We examined EMT markers, migration and invasion assays in survivin disrupted ovarian cancer cells using Transwell plates. We also assessed primary ovarian tumor growth and metastasis in an orthotopic ovarian cancer mouse models through intrabursal injecting survivin KO cell line and also treatment using MX106. Findings: We found that disruption of survivin expression inhibited EMT by attenuating the TGFβ pathway in ovarian cancer cells. Cell proliferation, migration, and invasion were significantly inhibited in survivin knockout or MX106 treatment in SKOV3 and OVCAR3 cell lines compared to control cells. Inhibition of survivin sensitized cell responses to paclitaxel treatment and overcame chemoresistance in vitro. Lentiviral CRISPR/Cas9 mediated survivin knockout or MX106 treatment efficiently inhibited primary ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models. Conclusions: Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis by attenuating TGFβ pathway. MX106, as a novel survivin inhibitor effectively overcame chemoresistance compared to commercial YM155, which provides a novel approach for ovarian cancer therapy. Citation Format: Junming Yue. Genetic and pharmacological disruption of survivin suppressed primary and metastatic tumor growth by attenuating TGFB pathway in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4184.

Background: Previously we found that survivin is expressed on the surface of cancer cells in vitro, and is targetable using certain survivin-specific antibodies. The survivin gene is alternatively spliced into two alternative isoforms, ΔEX3 and 2B, which diverge structurally from the canonical isoform in their C-termini but retain a conserved BIR (Baculovirus IAP Repeat) domain. Methods: In order to characterize plasma membrane survivin (PM-SVN), tagged proteins representing canonical survivin (isoform 1), Δex3 and 2B were transiently expressed in multiple cancer cell line backgrounds. Relative surface expression was measured by flow cytometry staining of intact cells. Mutant constructs were used to determine the structural requirements for translocation. Imaging flow cytometry (Imagestream) was used to visualize survivin isoform localization, and to detect co-localization on the cell surface. Secreted exosomes were examined for expression of survivin isoforms. Results: Isoforms 1, Δex3 and 2B could be detected on the surface of HEK293T embryonic kidney, HeLa cervical adenocarcinoma, U87 glioblastoma, and A1207 glioblastoma cells. PM-SVN expression was variable, however Δex3 showed robust membrane expression across all cell lines. Deletion of a predicted transmembrane sequence in Δex3's unique C-terminus did not impair its translocation, and deletion of the first 10 amino acids, thought to be required for dimerization, disrupted surface expression only in HEK293T cells. Imaging flow cytometry analysis of cells revealed polar localization of PM-SVN, with specific punctate areas seen in 13-20% of cells. Staining with antibodies targeting both isoform 1 and Δex3 showed co-immunofluorescence in cell membrane puncta, indicating the formation of heterodimers. Stressing cells by heat shock increased the proportion of PM-SVN in 293T and A1207 cells; in 293T, this was accompanied by enrichment of survivin protein in secreted exosomes. Conclusions: In addition to canonical survivin, isoforms Δex3 and 2B are displayed on the surface of cancer cells, and can be secreted as exosomal cargo. Membrane translocation may be stress-induced and appears to require the conserved BIR domain. PM-SVN shows a polar distribution on the cell membrane, and isoforms 1 and Δex3 heterodimerize within these puncta. Citation Format: Sheila Figel, Meaghan Birkemeier, Sanam Dharma, Emma Steinmetz, Michael Ciesielski, Robert Fenstermaker. Canonical and noncanonical survivin isoforms translocate to the plasma membrane and are secreted in exosomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2939.

Metastasis is a major cause of death in epithelial cancers, largely because of a lack of effective treatments. The metastatic inhibitor MBQ-167 was developed to target the Rho GTPases Rac and Cdc42, which promote cell cycle progression, proliferation, survival, migration/invasion, and thus, metastasis. Our published data show that MBQ-167 inhibits the activation of Rac and Cdc42 with ~100 nM IC50, and subsequently inhibits their effector p21-activated kinase (PAK) activation by autophosphorylation. In metastatic cancer cells, MBQ-167 induces a loss of cell polarity, followed by detachment from the substrate, and subsequent apoptosis (anoikis). The objective herein was to determine the potential off-target effects of MBQ-167. Therefore, the effect of MBQ-167 on the triple negative breast cancer kinome was investigated using a Human Phospho-Kinase Array for 45 phospho proteins. MDA-MB-231 cells treated with MBQ-167 for 24 h at 200 nM demonstrated no difference in phosphorylation of off-target kinases. However, phospho (p)-CREB and p-c-Jun transcription factor levels increased by ~3-fold in the cells that remained attached following MBQ-167 treatment. This was confirmed by Western blot for p-CREB and p-c-Jun as well as their transcriptional targets (Cyclin D1, survivin, ZEB1). However, a time-course analysis via Western blot revealed that this increase in CREB and c-Jun activity is transient. Western blotting of the attached and detached cell populations of MDA-MB-231 cells following MBQ-167 treatment for 24, 48, or 96 h demonstrated decreased c-Jun/p-c-jun, and CREB and Jun targets in the detached cell population. Moreover, the detached cells demonstrated additional decreases in phospho forms of ERK1/2, Akt, mTOR, beta catenin and STAT3, which have all been implicated in Rac/PAK signaling; as well as reduced expression of the anti-apoptotic proteins Bcl2, BCL-XL, and MCl-1. Therefore, we posit that the initial upregulation of transcription factors in response to MBQ-167 is a transient reaction to its strong Rac/Cdc42/PAK inhibition. Prolonged treatment with MBQ-167 causes a massive deregulation of cancer drivers and pro-survival proteins to ultimately result in anoikis. We attempted to create a MBQ-167 resistant cell population by treating MDA-MB-231 cells with ascending concentrations of MBQ-167 over time, but were unable to recover any live cells. Therefore, we conclude that MBQ-167 is a direct and specific inhibitor of Rac and Cdc42 and that potential off-target effects of MBQ-167 are only temporary, where 100% of the metastatic cancer cells treated with MBQ-167 ultimately undergo apoptosis. Collectively, our data on the mechanism of action of MBQ-167 and its efficacy in vitro and in vivo in mouse models of metastatic cancer demonstrate that MBQ-167 is a viable anti-metastatic cancer inhibitor for further development as an experimental therapeutic. Citation Format: Hector M. Picon, Maria Del Mar Maldonado, Surangani F. Dharmawardhane Flanagan. Investigating potential off-target effects of the dual Rac and Cdc42 inhibitor MBQ-167 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 623.

The serine/threonine kinase LKB1 is a well-characterized tumor suppressor that governs diverse cellular processes, including growth, polarity, and metabolism. Somatic-inactivating mutations in LKB1 are observed in about 15% to 30% of non–small cell lung cancers (NSCLC). LKB1 inactivation confers lung adenocarcinomas (ADC) with malignant features that remain refractory to therapeutic intervention. YAP activation has been linked to LKB1 deficiency, but the role of YAP in lung ADC formation and progression is uncertain. In this study, we showed that ectopic expression of YAP in type II alveolar epithelial cells led to hyperplasia in mouse lungs. YAP overexpression in the KrasG12D lung cancer mouse model accelerated lung ADC progression. Conversely, YAP deletion dramatically delayed the progression of lung ADC in LKB1-deficient KrasG12D mice. Mechanistic studies identified the antiapoptotic oncoprotein survivin as the downstream mediator of YAP responsible for promoting malignant progression of LKB1-deficient lung ADC. Collectively, our findings identify YAP as an important contributor to lung cancer progression, rationalizing YAP inhibition in the context of LKB1 deficiency as a therapeutic strategy to treat lung ADC. Cancer Res; 75(21); 4450–7. ©2015 AACR.

Infection of T cells with human T-cell leukemia virus type-1 (HTLV-1) induces clonal proliferation and is closely associated with the onset of adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. Although Tax expression is frequently suppressed in HTLV-1-infected cells, the accessory gene, HTLV-1 bZIP factor (HBZ), is continuously expressed and has been implicated in HTLV-1 pathogenesis. Here, we report that transduction of mouse T cells with specific mutants of HBZ that distinguish between its RNA and protein activity results in differential effects on T-cell proliferation and survival. HBZ RNA increased cell number by attenuating apoptosis, whereas HBZ protein induced apoptosis. However, both HBZ RNA and protein promoted S-phase entry of T cells. We further identified that the first 50 bp of the HBZ coding sequence are required for RNA-mediated cell survival. Transcriptional profiling of T cells expressing wild-type HBZ, RNA, or protein revealed that HBZ RNA is associated with genes involved in cell cycle, proliferation, and survival, while HBZ protein is more closely related to immunological properties of T cells. Specifically, HBZ RNA enhances the promoter activity of survivin, an inhibitor of apoptosis, to upregulate its expression. Inhibition of survivin using YM155 resulted in impaired proliferation of several ATL cell lines as well as a T-cell line expressing HBZ RNA. The distinct functions of HBZ RNA and protein may have several implications for the development of strategies to control the proliferation and survival mechanisms associated with HTLV-1 infection and ATL. Cancer Res; 75(19); 4143–52. ©2015 AACR.

ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway–inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses. Cancer Res; 75(8); 1668–74. ©2015 AACR.

Glutamate-triggered signal transduction is thought to contribute widely to cancer pathogenesis. In melanoma, overexpression of the metabotropic glutamate receptor (GRM)-1 occurs frequently and its ectopic expression in melanocytes is sufficient for neoplastic transformation. Clinical evaluation of the GRM1 signaling inhibitor riluzole in patients with advanced melanoma has demonstrated tumor regressions that are associated with a suppression of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways. Together, these results prompted us to investigate the downstream consequences of GRM1 signaling and its disruption in more detail. We found that melanoma cells with enhanced GRM1 expression generated larger tumors in vivo marked by more abundant blood vessels. Media conditioned by these cells in vitro contained relatively higher concentrations of interleukin-8 and VEGF due to GRM1-mediated activation of the AKT–mTOR–HIF1 pathway. In clinical specimens from patients receiving riluzole, we confirmed an inhibition of MAPK and PI3K/AKT activation in posttreatment as compared with pretreatment tumor specimens, which exhibited a decreased density of blood vessels. Together, our results demonstrate that GRM1 activation triggers proangiogenic signaling in melanoma, offering a mechanistic rationale to design treatment strategies for the most suitable combinatorial use of GRM1 inhibitors in patients. Cancer Res; 74(9); 2499–509. ©2014 AACR.

Wnt signaling is a critical mechanism of biological processes, including cell proliferation, tissue homeostasis and development. The aberrant activity of Wnt signaling pathway is highly correlated with the colorectal carcinogenesis. Therefore, the effective compounds targeting Wnt signaling pathway could be applicable for colorectal cancer treatment. The bioactivity of nodosin, a component of Isodon serra, was evaluated in HCT116 colorectal cancer cells. Nodosin exhibited a potential growth inhibition of colon cancer cells. Nodosin also showed an effective inhibition upon wnt/β-catenin reporter gene assay in HEK293 and HCT116 cells. In addition, Wnt target genes such as β-catenin, Axin2, cyclin D1 and survivin were suppressed by nodosin in HCT116 cells. The inhibition of cell proliferation by nodosin was in part associated with the G2/M phase cell cycle arrest in colon cancer cell. These findings suggest that the anti-proliferative activity of nodosin against colorectal cancer cell should be associated with the regulation of Wnt/β-catenin signaling pathway. This work was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF-2016M3A9B6903499). Citation Format: Eunseo Bae, Donghwa Kim, Woong sub Byun, Sang Kook Lee, Young-Won Chin. Anti-proliferative activity of nodosin is associated with the regulation of wnt signaling pathway in colon cancer cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4304.

Background: Despite the advent of HER2-targeted therapies, including the monoclonal antibody trastuzumab (T) and the HER1/2 inhibitor lapatinib (L), for HER2+ breast cancer (BC), resistance still poses a major challenge. L and L+T resistant (R) HER2+ cells with inhibited HER2 signaling showed upregulation of RNA levels of the mevalonate pathway (MVA) enzymes (which were inhibited by short term treatment of parental (P) cells with L or L+T) and increased sensitivity to MVA inhibition by statins (e.g. simvastatin (Sim)), suggesting MVA’s role as an escape mechanism of resistance. Here we investigated the therapeutic potential of another MVA inhibitor Zoledronate (ZA) and the role of mTOR and YAP/TAZ (Y/T) in mediating this resistance. Lastly, we tested if co-blockade of the MVA or its downstream effectors further sensitizes HER2+ models to anti-HER2 therapies. Methods: SKBR3 and AU565 P cells and their LR and LTR derivatives were used. The effects of MVA perturbations on cell growth and HER2 or MVA signaling were studied by methylene blue staining and Western blot (WB), respectively. Y/T activity was tested by a luciferase reporter assay and functionally validated by siRNA knockdown and dominant-active (DomA) YAP overexpression. YAP target gene expression was assessed by RT PCR. SCID-Beige mice bearing HER2+ BCM-3963 PDX tumors were treated with vehicle, L, Sim, or L+Sim and monitored for time to complete response (CR). Results: ZA, like Sim, showed a selective inhibition of cell growth and mTOR signaling in R vs. P cells, which was rescued only by the downstream metabolite geranyl geranyl pyrophosphate (GGPP), but not by the upstream metabolite mevalonate, indicating the on-target effect of ZA. Increased Y/T activity in R models was confirmed, and both Sim and ZA inhibited TAZ levels and induced phospho-YAP levels, which were rescued by the corresponding downstream metabolites. Y/T knockdown inhibited growth and mTOR signaling in R vs. P cells, and DomA YAP negated the mTOR inhibition by Sim. Sim and ZA also significantly decreased levels of the Y/T target gene survivin in R vs. P cells, and the expression was rescued by the downstream metabolites. Inhibition of MVA by Sim or ZA or its downstream signaling effectors, Y/T (by siRNA) and mTOR (by everolimus), enhanced the L sensitivity in P cells. Conversely, DomA YAP reduced the sensitivity of P cells to L. In the presence of Sim or ZA, L treatment more strongly inhibited levels of phospho-S6, a downstream target of mTORC1, compared to L alone. Preliminary in vivo data showed that treatment with L+Sim vs. L alone shortened the median time to CR and numerically increased CR rates. Conclusions: The MVA pathway mediates anti-HER2 therapy resistance via Y/T, survivin, and mTOR, in some cell models and this resistance can be overcome by Sim and ZA. The potential of MVA pathway inhibition to enhance anti-HER2 therapy efficacy warrants further clinical studies. Citation Format: Vidyalakshmi Sethunath, Huizhong Hu, Carmine DeAngelis, Jamunarani Veeraraghavan, Lanfang Qin, Martin Shea, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Susan G. Hilsenbeck, Mothaffar F. Rimawi, Kent C. Osborne, Rachel Schiff. Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4757.

Anti-apoptotic B cell lymphoma 2 (Bcl-2) family proteins are frequently overexpressed across a variety of tumors, resulting in tumor cell survival and resistance to therapy. Inhibition of the expression or activity of these survival factors is an attractive approach for cancer therapy. Activating transcription factor 5 (ATF5) regulates gene transcription of anti-apoptotic Bcl-2 family proteins in neural progenitor cells and a wide range of human cancer cells. Here, we describe ST101, a rationally designed, synthetic, D-amino acid, cell penetrating peptide therapeutic designed to disrupt the protein-protein interactions driving ATF5-regulated gene transcription. Exposure of HL60 promyelocytic leukemia cells and MCF7 breast adenocarcinoma cells to low micromolar concentration of ST101 resulted in a decrease in MCL-1, BCL-2 and BIRC5 (Survivin) mRNA expression at 4 and 24 hrs post exposure. Further, exposure to ST101 resulted in a dose-dependent loss of viability across a panel of human cancer cells, including MCF7, HL60, U251 glioblastoma, A375 melanoma, DU145 prostate cancer, and A549 lung adenocarcinoma, characterized by an increase in annexin V and PI staining by flow cytometry peaking 48 hrs post exposure, resulting in a median half maximal effective concentration (EC50) value of 4.0 micromolar. In contrast, normal human peripheral blood mononuclear cells and bone marrow mononuclear cells were resistant to ST101-mediated cell death, with >80 micromolar EC50 values. In mouse xenograft experiments, 25mg/kg ST101 administered three times per week for three weeks resulted in significant tumor regression in MCF7 and U251 subcutaneous tumors as well as tumor growth delay in HL60 subcutaneous tumors. Tumor growth remained significantly inhibited weeks after the last treatment in the MCF7 and U251 models. In summary, ST101 selectively kills cancer cell lines by decreasing BCL-2 family gene expression, resulting in significant reductions in tumor growth in mouse models. Taken together, these data validate ST101 as a potent peptide therapeutic candidate for a variety of solid tumor and hematologic malignancies. Citation Format: Jim A. Rotolo, Rick Ramirez, Mark Koester, Siok Leong, Lila Ghamsari, Gene Merutka, Barry J. Kappel. Cell penetrating peptide, ST101, disrupts ATF5 regulation of anti-apoptotic Bcl-2 family proteins, resulting in induction of cancer cell death in vitro and tumor growth inhibition/regression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2050.

Drug repositioning can benefit from reduced development risk in terms of safety, toxicity and dosage, as it centers upon drugs that have already been tested in clinical trials. Disulfiram (DSF), a drug for the treatment of alcoholism, has potential anti-cancer activities in a copper (Cu)-dependent manner. In the present study, we investigated the effect of DSF on cell viability, apoptosis, breast cancer stem cells (BCSC)-like properties and p53 activation in ER-positive breast cancer MCF7 and T47D cells. DSF treatment caused a significant suppression of cell viability and marked induction of cell death in a Cu-dependent manner. This response was accompanied by caspase-3/-7 and p53 activation in ER-positive breast cancer cells. The latter phenomenon was associated with G1 phase arrest, coinciding with a significant increase in accumulation of nuclear p21 and downregulation of cyclin D1 and survivin. DSF treatment elicits a significant inhibitory effect on these BCSC-like properties including ALDH1 activity and CD44+/CD24- subpopulations. To examine the effect of DSF on mammosphere-forming ability, MCF7 and T47D cells were cultured in anchorage-independent serum-free culture conditions in the presence or absence of DSF and Cu. DSF considerably suppressed mammosphere formation, as evidenced by significant reduction in the number and volume of mammospheres. Our results demonstrate that DSF induces apoptosis and effectively targets cancer stem-like population, suggesting that disulfiram may be potentially effective for the treatment of ER-positive cancer. Citation Format: Seojin Jang, Eunhye Oh, Yoon-Jae Kim, Tae-Min Cho, Jung Min Park, Soeun Park, Minsu Park, Jae Hong Seo, Ji Young Kim. Disulfiram targets both proliferating cancer cells and cancer stem-like population in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1152.

Breast cancer is one of the most common malignancies and the second leading cause of cancer related mortality among women in U.S., thus developing new strategies to control breast cancer is an important mission. Repurposing of old drugs as new anti-cancer drugs is important as it can save time and cost of drug development. Proguanil is used in combination with atovaquone for the treatment of malaria. In this study, we determined the anticancer effects of proguanil in breast cancer cells. Proguanil significantly reduced the viability of MDA-MB-231, HCC1806 and MCF-7 breast cancer cells with IC50 of 42μM, 44μM and 40μM respectively after 72 h of treatment and induced apoptosis as exhibited by FITC/Annexin assay and cleavage of caspase 3 as well as PARP. Proguanil also reduced the survival of several patient-derived cells with IC50 in the range of 30-40μM. The anti-cancer effects of proguanil were associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. ROS generation by proguanil was about 2-3 fold more as compared to control in MDA-MB-231 and HCC1806 cells. The generation of ROS was inhibited when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Moreover, exposure of MDA-MB-231 and HCC1806 cells to proguanil was also associated with increased expression of Bax, p-H2AX, c-caspase9 and down-regulation of bcl-2 and survivin. We further evaluated the oxygen consumption rate with proguanil treatment in breast cancer cells using flux analyzer. Our results showed significant decrease in the mitochondrial respiration rate and ATP production rate after proguanil treatment. Furthermore, 20mg/kg proguanil when given orally suppressed the growth of 4T1 breast tumors in female Balb/c mice by 55%. Tumors from proguanil treated mice demonstrated increased apoptosis, which was related to the increased expression of p-H2AX and Bax. Taken together, these results show that proguanil is an effective inhibitor of in vitro and in-vivo growth of breast cancer cells. These findings provide the rationale for further clinical investigation of proguanil against breast cancer. Citation Format: Nehal Gupta, Sanjay Srivastava. Oxidative stress by proguanil suppresses breast tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4795.

Background: Enzalutamide is an FDA-approved drug commonly prescribed for advanced prostate cancer. Enzalutamide slows down prostate tumor growth but resistant disease invariably develops which is incurable primarily because currently available therapies cannot effectively kill enzalutamide-resistant prostate cancer cells. Mechanism(s) behind development of enzalutamide-resistance is not properly understood, though overactivation of c-Myc has been found to be a common event which plays an important role in the maintenance and progression of ERPC phenotype. However, direct-targeting of c-Myc poses special problem because of its non-enzymatic nature and certain amount of c-Myc activity is needed by non-cancer cells as well. Thus, c-Myc has emerged as an elusive target which needs to be managed by novel agents and strategies in a cancer-specific way for better control of ERPC. Methods: We addressed this problem by treating ERPC cells with a variety of cell survival and apoptosis-regulating agents followed by measurement of cell viability, and by analysis of the mRNA and protein levels of c-Myc. Gene expression was analyzed by Illumina Hi-Seq whole genome gene-expression array. Expression of c-Myc was confirmed by RT-PCR and Western blot. Apoptosis was measured by annexin-V binding, PARP-cleavage, and by detecting degradation of chromatin-DNA to nucleosomes. Transcriptional activity of c-Myc was analyzed by nuclear accumulation, DNA-binding, luciferase-reporter assays and expression of c-Myc-target genes. Results: We found that MK591, a leukotriene biosynthesis inhibitor, dramatically downregulates c-Myc in ERPC cells as revealed by RT-PCR, and Western blot as well as by the reduction in nuclear-accumulation and DNA-binding activities of c-Myc. Treatment with MK591 decreased the Myc-driven E-box-luciferase reporter activity, and substantially reduced the expression of c-Myc target genes (Cyclin D1, CDK4, survivin, Aurora kinase). Moreover, MK591 effectively blocked in vitro invasion and soft-agar colony-formation by ERPC cells. Interestingly, while MK591 strongly inhibits c-Myc function and kills ERPC cells via caspase-mediated apoptosis, it does not inhibit the basal c-Myc function or the viability of non-cancer cells, such as human foreskin fibroblasts (HFF). Conclusion: Our findings indicate that the expression and oncogenic-function of c-Myc in ERPC cells are severely downregulated by MK591, and suggest that MK591 may turn out to be a suitable new agent to treat advanced, aggressive prostate cancers which are resistant to enzalutamide therapy. Citation Format: Jitender Monga, Ajay Bharathan, Dhatchayini Subramani, Jagadananda Ghosh. MK591 (Quiflapon) downregulates c-Myc oncogenic signaling and induces apoptosis in enzalutamide-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1275.

We previously reported that phosphodiesterase 10A (PDE10) is overexpressed during early stages of lung cancer and is essential for lung tumor cell growth. Here we characterize a novel PDE10 inhibitor, MCI-048, that was identified by screening a library of indene compounds. MCI-048 potently inhibited the growth and induced apoptosis of multiple human lung cancer cell lines expressing PDE10, while normal human airway epithelial cells lacking PDE10 expression were appreciably less sensitive. The mechanism of action of MCI-048 involves PDE10 inhibition, cGMP elevation, PKG activation, and phosphorylation of β-catenin at key residues that induce ubiquitination and proteosomal degradation of the oncogenic pool of β-catenin in cytoplasm to suppress the translocation of active β-catenin to the nucleus and Lef/Tcf-mediated transcription of genes encoding for proteins such as c-myc, cyclin D, and survivin, which are essential for tumor cell proliferation and survival. Pharmacokinetic and tissue distribution studies revealed a unique feature of MCI-048 to accumulate at high concentrations in lungs relative to plasma and other tissues. To assess the potential of MCI-048 for the treatment of lung cancer and blocking metastasis, we tested the drug in two mouse models of lung cancer involving either orthotopic implantation of KRAS mutant A549 lung tumor cells or the chemical carcinogen, urethane. Oral administration of MCI-048 significantly inhibited tumor growth and extended survival in the orthotopic model using two different protocols to either treat the primary tumor or metastasis. Similarly, MCI-048 significantly reduced tumor burden as measured by both surface counting and histopathological analysis in the urethane-induced model of lung tumorigenesis. Biochemical analysis showed that MCI-048 reduced levels of urethane-induced active Ras-GTP and PDE10 levels, as well as other oncogenic markers, including pEGFR and c-Myc. Both mouse models revealed that MCI-048 was well tolerated with no discernable toxicity, thus supporting preclinical development for the treatment of lung cancer. Funding provided by NCI grants R21CA182941, R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Bing Zhu, Veronica Ramirez-Alcantara, Antonio Ward, Kristy Berry, Adam B. Keeton, Michael R. Boyd, Yulia Maxuitenko, Xi Chen, Gary A. Piazza. A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2731.

Over 90% of colorectal cancers harbor mutations in β-catenin or pathway components (e.g. APC) that stabilize β-catenin, causing nuclear translocation and constitutive Tcf-mediated transcription of genes encoding proteins essential for the proliferation and survival of tumor cells. We recently reported that the cyclic nucleotide degrading phosphodiesterase (PDE) isozyme PDE10 is overexpressed in colorectal cancers relative to normal tissue. Its expression and enzymatic activity are essential for colon tumor cell growth, as evidenced by knockdown of PDE10 expression using siRNA or inhibition of enzyme activity using known inhibitors such as PF-2545920. PDE10 inhibition in tumor cells expressing high levels of PDE10 causes increased intracellular cGMP levels to activate PKG and phosphorylate β-catenin, which induces ubiquitination and proteasomal degradation to suppress nuclear translocation and Tcf transcriptional activity. Conversely, ectopic expression of PDE10 in normal colonocytes or precancerous adenoma cells causes increased levels of β-catenin and the expression of proteins (e.g. cyclin D and survivin) essential for the proliferation and survival of tumor cells. To identify novel antitumor PDE10 inhibitors, we screened a chemically diverse library of indenes for PDE10 and tumor cell growth inhibitory activity. Following extensive chemical optimization, MCI-030 emerged as a potent and selective inhibitor of tumor cell growth. Similar to PF-2545920, but with appreciably greater potency and tumor cell selectivity, MCI-030 inhibited colon tumor cell growth by activating cGMP/PKG signaling to phosphorylate and induce β-catenin degradation. MCI-030 also inhibited colon tumor cell spheroid formation and reduced spheroid size and growth at concentrations that inhibit PDE10. Oral administration of MCI-030 significantly inhibited colon tumor formation in the Apc+/min-FCCC mouse model without discernable toxicity. Importantly, unlike PDE10 inhibitors developed to cross the blood-brain barrier for the treatment of CNS disorders, MCI-030 lacks the sedation side effects. Together, these findings support preclinical development of MCI-030 for the treatment of colorectal cancer as a novel PDE10 inhibitor capable of selectively inhibiting the growth of tumors harboring β-catenin or APC mutations. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Antonio B. Ward, Xi Chen, Jacob Valiyaveettil, Kevin Lee, Wen-Chi L. Chang, Yulia Maxuitenko, Veronica Ramirez-Alcantara, Kristy Berry, Luciana Madeira da Silva, Bing Zhu, Tyler Mattox, Michael R. Boyd, Adam B. Keeton, Margie L. Clapper, Harry S. Cooper, Gary A. Piazza. A novel PDE10/β-catenin pathway inhibitor, MCI-030, for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3864.

Introduction. Medulloblastoma is the most common malignant brain tumor in children. New treatment strategies are urgently needed as the current options often result in significant long term neurocognitive and growth defects among survivors. We have previously demonstrated the anti-cancer activity of small-molecule non-steroidal anti-inflammatory drug tolfenamic acid (TA) in medulloblastoma cells and tumor model. The anti-cancer activity of TA can be correlated to its ability to downregulate the anti-apoptotic protein Survivin, cause cell cycle arrest, and induce apoptosis. A recent publication has shown that the activity of TA can be enhanced by forming a complex with copper (II). In this study we tested the function of the copper complex as an anti-cancer agent against pediatric medulloblastoma. Methods. Pediatric medulloblastoma cell lines DAOY and D283 were used in this study. We have previously demonstrated the synthesis, stability, and anti-cancer activity of Copper-Tolfenamic acid (Cu-TA) complex using various cancer cell lines. The efficacy of Cu-TA against medulloblastoma cells was determined by studying the effect of increasing dose on cell viability, using the CellTiter-Glo Luminescent Cell Viability Assay. Further experiments, using optimized doses of Cu-TA, were carried out to determine its effect on cell cycle and its ability to induce apoptosis. Based on our previous results with TA, we also investigated the effect of Cu-TA on survivin protein levels. Results. Inhibition of cell viability was observed with increasing doses of Cu-TA as well as with increasing time. The IC50 values were 2-fold lower compared to that of TA and were used in further experiments. Cell cycle analysis using propidium iodide staining and flow cytometry revealed that treatment with Cu-TA results in accumulation of cells in G0/G1 phase, as early as 12h after treatment. Cu-TA induced apoptosis was studied by flow cytometric analysis of AnnexinV stained cells and by western blot analysis of cleaved-PARP. At 48h post-treatment, significant increase in apoptosis was detected by flow cytometry, that also correlated with increased levels of c-PARP. Cu-TA treatment also resulted in a significant decrease in survivin levels compared to TA at 24 and 48h post-treatment. Conclusion. Our earlier studies with TA have demonstrated its potential as an anti-cancer agent with relatively safe toxicity profile, both in vitro and in pre-clinical mouse model studies. TA also has the capability to enhance the response of standard treatments when used in combination. Here, we demonstrate the ability to increase the efficacy of TA when complexed with copper (II). Cu-TA, with its increased anti-cancer activity and potential to sensitize cells to chemotherapy and radiation, provides a novel therapeutic option for the treatment of medulloblastoma. Citation Format: Julie Hong, Melissa Schullek, John K. Smith, Joseph Reitman, Amil Dudhia, Nihant Tallapaka, Rassil Basha, Deondra T. Brown, Jaya Chhabra, Alvin Holder, Umesh T. Sankpal. Effect of novel copper-tolfenamic acid complex on medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2972.

Treatment with bromodomain and extra-terminal protein inhibitor (BETi) reduces in vivo AML cell burden, inducing clinical remissions in AML. Yet, resistance to BETi treatment develops uniformly. Here, following at least 10 exposures of secondary (s) AML control (parental) SET2 and HEL92.1.7 cells to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we generated BETi persister-resistant (BETi-P/R) SET2-P/R and HEL-P/R cells. These cells showed > 10-fold resistance to OTX015 and cross-resistance to other BETis. Compared to the controls, SET2-P/R and HEL-P/R cells lacked additional genetic alterations or altered levels of TRIM33, SPOP, DUB3 or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). However, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels of β-catenin, the transcription factor TCF7L2 and adaptor protein TBL1X (TBL1), associated with increased expression of TCF7L2 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, with enrichment of STAT5, MYC, PU.1 and GATA2 binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with induction of gene-sets involving MYC/MAX, STAT5, NFkB and TCF7L2 targets. QPCR and Western analyses confirmed increase in the mRNA and protein levels of TCF7L2, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Also, confocal microscopy demonstrated increased binding of β-catenin with TBL1 and TCF7L2 in the nucleus in BETi-P/R sAML cells. β-catenin inhibitor BC2059, which disrupts binding of nuclear β-catenin with TBL1 and TCF7L2 and depletes β-catenin levels, exerted similar lethality in BETi-P/R sAML and control sAML cells. Both shRNA-mediated knockdown of BRD4 and BRD4-PROTAC (proteolysis-targeting chimera) ARV-771 (Arvinas, Inc.), which degrades BRD4/3/2, induced similar level of apoptosis in BETi-P/R and control sAML cells. Co-treatment with ARV-771 and BC2059 synergistically induced lethality in BETi-P/R sAML cells as well as in patient-derived, CD34+ sAML BPCs (combination indices < 1.0). This was associated with marked attenuation of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL levels. Also, compared to each agent alone, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW, per week) for 3 weeks, significantly reduced sAML burden and improved survival of NSG mice engrafted with HEL-P/R cells (p < 0.01). Thus, increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BRD4 degrader and β-catenin-TCF7L2 inhibitor is a promising therapeutic strategy against BETi-P/R sAML BPCs. Citation Format: Dyana T. Saenz, Warren C. Fiskus, Taghi Manshouri, Christopher P. Mill, Steve Horrigan, Raffaella Soldi, Joseph D. Khoury, Sunil Sharma, Srdan Verstovesek, Kapil N. Bhalla. BRD4 degrader and inhibitor of beta catenin-TCF7L2 are synergistically active against human AML cells resistant to BET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3036.

Osteosarcoma is one of the most prevalent malignant bone tumors in children and adolescents and has the characteristics of early metastasis, rapid progression and poor prognosis. Although with the advancement in techniques and treatment increasing the survival rates up to 60%, the prognosis remains poor for most patients with metastatic or recurrent osteosarcoma. Identifying the molecular mechanism and developing therapeutic agents for metastasis is urgent. Aurora B kinase play a key role in mitosis and have been reported to be associated with cancer metastasis. However, the role of Aurora kinase in osteosarcoma is still unclear. In this study, we identified a novel compound, referred to as APIO-EE-7, which inhibits growth and colony formation and induces apoptosis in osteosarcoma. Using computer modeling, we found that APIO-EE-7 can interact with Aurora B at ATP-binding pocket. Treatment with APIO-EE-7 can increase EMT marker expression level of E-cadherin and decrease N-cadherin, vimentin and survivin expression. Importantly, APIO-EE-7 dramatically attenuated tumor incidence and volume in osteosarcoma metastasis mouse model. Our findings suggest that APIO-EE-7 is a potential therapeutic agent against osteosarcoma metastasis by targeting Aurora B, which provides hope for rapid clinic translation. Citation Format: Guoguo Jin, Jitian Li, Zhiping Guo, Junyan Teng, Xiaoling Li, Zhenjiang Zhao. Molecular mechanism of APIO-EE-7 suppresses cell metastasis by targeting Aurora B in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4292.

The development of inhibitors of Signal transducer and activator of transcription (STAT) 3 for clinical application has faced significant challenges and to date there are no suitable STAT3 small molecule inhibitors in the clinic. We report a novel class of azetidine-based small molecules that potently and preferentially inhibit STAT3 DNA-binding activity in vitro, with IC50 of 300-800 nM, compared to IC50 of 17 μM or higher against STAT1 DNA-binding activity. In the human breast cancer MDA-MB-231 and MDA-MB-468 cells treated with the azetidine compounds, including H182, constitutive STAT3 DNA-binding activity and tyrosine phosphorylation were strongly inhibited in both time- and dose-dependent manner. By contrast, the azetidine compounds showed little effects on EGF-induced STAT1 activity, Ras-Raf-MEK-ERK or other STAT3-independent signaling pathways, or on other SH2 domain-containing proteins. Furthermore, H182 and other azetidine compounds strongly inhibited viability, anchorage-dependent and -independent growth, and colony survival of the human breast cancer cells, with IC50 of 1.0 - 1.9 μM, compared to the IC50 of 3.8 - 8.1 μM against the viability and growth of the immortalized breast epithelial MCF-10A cells. The migration of breast cancer cells in vitrowere similarly inhibited by treatment with low concentrations of H182 for 18 h, without changes in viable cell number. Analysis of STAT3 downstream genes show that the expression of VEGF and survivin were significantly suppressed in breast cancer cells treated with the azetidine compounds. In vivo evaluation of antitumor efficacy shows H182 inhibited growth of human breast tumor xenografts. Studies together identify the azetidine-functionalized small molecules as potential candidate compounds for clinical development as novel therapeutic agents for human breast and other cancers that harbor aberrantly-active STAT3. Citation Format: Peibin Yue, Francisco Lopez-Tapia, Wenzhen Fu, Christine Brotherton-Pleiss, Marcus Tius, James Turkson. Azetidine functionalized small-molecules potently inhibit STAT3 signaling and block tumor growth in human breast cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4792.

Altered fatty acid metabolism has is a hallmark of cancer and continues to be a potential target for therapeutic intervention in cancer. Fatty Acid Translocase (CD36), a multifunctional glycoprotein, has been shown to have an important role in fatty acid metabolism as a fatty acid transporter. Fatty Acid Synthase (FASN), a critical enzyme involved in de novo lipogenesis, has been shown to be upregulated and associated with a poor prognosis in many cancers including colorectal cancer (CRC). However, the role of CD36 in CRC as well as its relation to de novo fatty acid synthesis is not understood. The purpose of our study was: (i) to determine the functional importance of CD36 in CRC and (ii) investigate potential relationships between CD36 and FASN expression in CRC cells. METHODS. Expression of CD36 and FASN was assessed by immunohistochemistry (IHC) using a CRC TMA with 2 sets of tissues: (i) Primary tumors with matched normal colon tissue; [n=56]; (ii) primary and metastatic tumors (liver [n=12] and lung metastasis [n=5]) with matched normal colon. Cellular proliferation was assessed in control and CD36 shRNA knockdown CRC cells, and in primary CRC cells established from patient derived xenografts treated with CD36 inhibitor Sulfo-N-succinimidyl oleate (SSO) in combination with FASN inhibitor TVB-3664. CD36 knockdown was confirmed by qRT-PCR. Expression of pro-survival and apoptotic markers was assessed via western blot in CD36 knockdown and overexpression CRC cells, as well as CD36+ and CD36- isogenic cells. CD36 localization was assessed via confocal imaging. RESULTS. We found that CD36 is overexpressed in primary tumors as compared to normal colon mucosa and its expression positively correlates with expression of FASN. Cellular proliferation was significantly reduced when CD36 was inhibited by SSO and a further reduction in proliferation was observed when SSO treatment was combined with TVB-3664. Knockdown and chemical inhibition of CD36 decreased expression of survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, which was shown to promote cancer cell survival in many tumor types. Conversely, overexpression of CD36 increased survivin expression in CRC cells. Additionally, shRNA knockdown of FASN induced CD36 expression in CRC cells. Immunofluorescence imaging of primary CRC cells treated with TVB-3664 showed an upregulation of membrane-bound CD36. CONCLUSION. Our studies indicate that CD36 upregulation is associated with an increase in cellular proliferation via upregulation of survivin in CRC cells. The correlation between CD36 expression and FASN suggest a connection between CD36 and de novo lipid synthesis. Furthermore, a decrease in FASN expression is associated with CD36 induction, suggesting a possible mechanism of resistance to FASN inhibition. Better understanding of the role of CD36 in CRC may provide new therapeutic approaches for treatment of CRC patients Citation Format: James Drury, Naser Jafari, B. Mark Evers Evers, Yekaterina Y. Zaytseva. Overexpression of CD36 promotes colorectal cancer cell proliferation via upregulation of survivin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4375.

Survivin is a protein expressed in a variety of human tissues whose function is to inhibit apoptosis. It achieves this by indirectly blocking caspase 3- 7- and 9- activation, thereby retarding or preventing programmed cell death. Survivin has been found to be highly expressed and specific in cancer tissues, but is absent in terminally differentiated cells, thus making it an attractive drug target. The structural basis through which survivin interacts with its neighboring proteins to inhibit apoptosis and promote cell growth is not well understood. Survivin is not believed to bind directly to caspases; instead, intermediary proteins such as XIAP, SMAC, and DIABLO are believed to first bind with survivin’s “BIR” domain and then bridge its binding to caspases. Unfortunately at the present, small molecule inhibitors against wild-type survivin have not demonstrated consistent anti-tumor activity to reach viability. The recent discovery of functional splicing variants for survivin (exon insertions or deletions) has drawn focus into whether these isoforms confer increased tumor resilience or aggressiveness, and whether inhibitors should specifically be designed for these targets. Specifically, the deltaEx3 (exon3 frameshift), 2B (intron 2 ins. of 69bp), 2alpha (intron 2 197bp tail ins.), and 3B (intron 3 165bp ins.) splice isoforms have been correlated with poor prognosis in AML, astrocytoma, breast, lung, renal, colorectal, brain, thyroid, bladder, prostate, sarcoma, and ovarian cancer. To date, no structural model, either crystal or NMR, has been elucidated for any of the survivin splice variants, nor has the full survivin molecule been solved in complex with its known ligands. We therefore employed a combination of homology modeling and molecular dynamics (MD) simulations to characterize and predict the three dimensional structure for the survivin’s most ubiquitous isoform, the 2B isomer, in which an alternative exon 2 termed “2B” includes a 69bp insertion. In addition, we characterized a model for survivin 2B complexed with SMAC/DIABLO, demonstrating for the first time in atomic level detail that the exon 2B insertion sequence plays a crucial role in stabilizing the survivin-SMAC/DIABLO interface involving both a hydrophobic interface protecting the binding pocket from solvent intrusion and also a conserved receptor-ligand network of stabilizing hydrogen bonds. By identifying the key residues and interactions on survivin’s BIR domain, we therefore can predict exposed regions which represent drug targets to competitively bind the BIR domain and prevent survivin 2B from initiating its functional cascade that inhibits apoptosis of malignant cells. The hypotheses generated from this study would naturally lend itself toward future high throughput studies for targeted small molecular inhibitors. Citation Format: Eric H. Lee, Chen-Shing Chen, Ly Le. Structural modeling and functional characterization of caspase inhibitor and tumor oncogene survivin 2B isoform: Implications in ligand binding and targeted drug design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3396.

Introduction/Purpose: Misregulation of BCL-2 family of proteins renders a survival signal to withstand cytotoxic anticancer drugs and is often found in drug resistant cells. The drug resistance phenotype often accompanies activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, as well as an enhancement of cancer stem cell-like (CSC) characteristics. Thus, inhibition of anti-apoptotic BCL-2 family proteins has been proposed as a possible antineoplastic strategy. However, the effects of BCL-2 inhibitors on drug resistant breast cancer have not yet been elucidated. In the present study, the effect of sabutoclax, a pan-active BCL-2 protein family antagonist, on two chemoresistant breast cancer cell lines was assessed. Methods: MTT assays and drug resistance clonogenic assays were performed to evaluate the cell viability in response to drug treatments. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays and the levels of a series of apoptosis-related proteins by real-time PCR and western blot.The expression levels of AKT and pAKT were assessed by western blot to exam the effect of sabutoclax on the activity of PI3K/AKT pathway. The expression of breast cancer stem cells (BCSC) related markers (CD44, CD24 and ALDH1) were detected by flow cytometry. Mammosphere formation assays and soft agar colony formation assay were performed to test the effect of sabutoclax on the proportion of stem-like cell population. To validate the results showed in breast cancer cell lines, we also tested the ex vivo effect of sabutoclax on nine fresh human breast tumor samples by IHC. The effect of sabutoclax on tumor growth was also studied in mouse xenografts. Results We found that sabutoclax showed a significant cytotoxic activity on chemoresistant breast cancer cells both in vitro and in vivo. When chemotherapeutic agents were combined with sabutoclax, strong synergistic antiproliferative effects were observed. Inhibition of BCL-2, MCL-1and BCL-xL leads to caspase-3/7 and caspase-9 activation, sabutoclax modulated Bax, Bim, PUMA and survivin expression. Furthermore, sabutoclax effectively eliminated the CSC subpopulation and reduced sphere formation of drug-resistant cells through down-regulation of the PI3K/AKT signaling pathway. A similar effect was observed in a panel of nine breast tumors ex vivo. Conclusions: Our findings indicate that sabutoclax partially overcomes drug resistance of breast cancer cells by reactivation of apoptosis, mediates by the inhibition of several anti-apoptotic BCL-2 family proteins such as BCL-2, MCL-1, BCL-XL and BFL-1, and, by abolition of PI3K/AKT pathway and crosstalk between AKT and BCL-2 family proteins is indispensable for maintaining stemness and chemoresistance in BCSCs. This suggests a strong rationale to explore the therapeutic strategy of using sabutoclax alone or in combination for chemotherapy-nonresponsive breast cancer patients. Citation Format: Zhang J. Sabutoclax, pan-active BCL-2 protein family antagonist, eliminates cancer stem cells through inhibiting PI3K/Akt pathway in breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-17.

Background: The HER2 pathway can be inhibited by potent targeting agents such as lapatinib (L), trastuzumab (T), or their combination (LT), but acquired and de novo resistance still occur. Resistance to these drugs remains a major hurdle in the management of HER2+ breast cancer. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER2-directed therapies is of critical importance. Methods: To obtain clues to the mechanisms for resistance we developed a panel of HER2+ breast cancer cell lines resistant to L, T, or LT. Parental cells and resistant derivatives of the HER2+ BT474 cell line were characterized by RNA-seq. Genes that were overexpressed in resistant compared to parental cells were confirmed by RT-PCR, Western blotting, and immunohistochemistry (IHC). Cell growth and cell signaling were assessed in parental and resistant cell lines after down-regulation (by siRNA) or overexpression (via an inducible cDNA) of IRS4 in the presence or absence of treatment. The effect of IRS4 overexpression on L resistance was assessed in a BT474 xenograft model. The proteins that interact with IRS4 were identified by co-immunoprecipitation with IRS4 followed by separation of the associated proteins by SDS-PAGE and microsequencing by mass spectrometry. Results: RNA-seq analysis revealed that IRS4 was the most up-regulated gene in BT474 L or LT resistant derivatives in which HER2 signaling is effectively inhibited, but not T alone, where HER2 signaling is reactivated. Western blotting and IHC validated this result and identified membrane localization of IRS4. Knockdown of IRS4 in L- or LT-resistant cells reversed resistance and restored growth inhibition. IRS4 knockdown also inhibited downstream signaling, with a reduction in pAKT but not in pMAPK. Induction of the cell cycle regulator p27 and down-regulation of survivin were observed after IRS4 knockdown. Overexpression of IRS4 cDNA in parental BT474 and SKBR3 cells led to resistance to L/LT, increased pAkt, and decreased the apoptotic marker cleaved PARP in the presence of L or the LT combination. The BT474 xenograft model showed that IRS4 overexpression in the absence of treatment had no effect on tumor growth but it significantly reduced the inhibitory effect of lapatinib (p=0.002). A group of proteins that interact with IRS4 in BT474 L-resistant cells were identified by mass spectrometry. The roles of these proteins in IRS4-mediated resistance to lapatinb-containing regimens are under investigation. Conclusion: IRS4 overexpression is a critical factor in causing resistance to lapatinib-containing regimens in BT474 cells. Investigation of IRS4 and its signaling partners in HER2+ human tumors resistant to lapatinib will be important to determine if this mechanism is also operative in patients. Citation Format: Lanfang Qin, Maria B Hahn, Xiaoyong Fu, Martin J Shea, Mario Giuliano, Sarmistha Nanda, Xiaowei Xu, Huizhong Hu, Sung Yun Jung, Laura M Heiser, Nicholas Wang, Joe W Gray, Susan G Hilsenbeck, Chad Creighton, Chad A Shaw, Gary C Chamness, Dean P Edwards, Sabrina Herrera, Carolina Gutierrez, C Kent Osborne, Rachel Schiff. Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-13.

The mTOR positively regulates cell proliferation and survival through forming 2 complexes with raptor (mTOR complex 1; mTORC1) or rictor (mTOR complex 2; mTORC2). Compared with the mTORC1, relatively little is known about the biologic functions of mTORC2. This study focuses on addressing whether mTORC2 regulates apoptosis, particularly induced by TRAIL (TNFSF10). Using the mTOR kinase inhibitor, PP242, as a research tool, we found that it synergized with TRAIL to augment apoptosis of cancer cells. PP242 reduced the abundance of the short form of c-FLIP (FLIPS, CFLARS) and survivin (BIRC5). Enforced expression of ectopic FLIPS, but not survivin, attenuated augmented apoptosis induced by PP242 plus TRAIL. Thus, it is FLIPS downregulation that contributes to synergistic induction of apoptosis by PP242 plus TRAIL. PP242 decreased FLIPS stability, increased FLIPS ubiquitination, and facilitated FLIPS degradation. Moreover, knockdown of the E3 ligase Cbl (CBL) abolished PP242-induced FLIPS reduction. Thus, PP242 induces Cbl-dependent degradation of FLIPS, leading to FLIPS downregulation. Consistently, knockdown of rictor or mTOR, but not raptor, mimicked PP242 in decreasing FLIPS levels and sensitizing cells to TRAIL. Rictor knockdown decreased FLIPS stability, whereas enforced expression of rictor stabilized FLIPS. Moreover, silencing of Cbl abrogated FLIPS reduction induced by rictor knockdown. Collectively we conclude that it is mTORC2 inhibition that results in FLIPS downregulation and subsequent sensitization of TRAIL-induced apoptosis. Our findings provide the first evidence showing that mTORC2 stabilizes FLIPS, hence connecting mTORC2 signaling to the regulation of death receptor-mediated apoptosis. Cancer Res; 73(6); 1946–57. ©2012 AACR.

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference–mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2+ tumors, potentially preventing acquired resistance to anti-HER2 therapy. Cancer Res; 73(3); 1190–200. ©2012 AACR.

Background: STAT3 has emerged as a novel potential anti-cancer target and its signaling is constitutively activated in various cancers including breast cancer. Our previous study has shown that STAT3 is a major kinase-independent target of sorafenib in HCC. (J Hepatol. 2011). Moreover, recent evidence has shown that p-STAT3 can be down-regulated by phosphatases, such as SHP-1 tyrosine phosphatase. We have designed and synthesized a series of sorafenib analogues devoid of sorafenib's kinase inhibition activity, some of which showed stronger p-STAT3 inhibition and apoptosis-inducing effects than sorafenib in HCC cells (Eur J Med Chem. 2011). Here, we tested the efficacy of novel sorafenib derivatives, SC-1 and SC-43, in breast cancer cells and examined the drug mechanism. Methods: Six Breast cancer cell lines were used for in vitro studies. Cell viability was examined by MTT-assay. Apoptosis was examined by both flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western Blot. SHP-1 activity was measured by a phosphatase activity assay kit. In vivo efficacy of Sorafenib, and sorafenib derivatives were tested in xenografted nude mice. Results: Sorafenib, SC-1 and SC-43 induced apoptosis in association with down-regulation of P-STAT3 and its downstream proteins Cyclin D1 and survivin in a dose-dependent manner in breast cancer cell lines (HCC-1937, MDA MB-468, MDA MB-231, MDA MB-453, SKBR-3, MCF-7) and the apoptotic effects induced by SC-1 and SC-43 were more potent than Sorafenib. Over-expression of STAT3 in MDA MB-468 cells protected cells from apoptosis induced by sorafenib, SC-1 and SC-43. Moreover, SC-1 and SC-43 up-regulated higher SHP-1 activity than sorafenib by in vitro phosphatase assays. Knockdown of SHP-1 by siRNA reduced apoptosis induced by SC-1 and SC-43. Importantly, SC-1 and SC-43 showed in vivo efficacy in MDA-468 xenografted tumor. Conclusions: Our data indicated that inhibiton of p-STAT3 by up-regulating SHP-1 activity mediated apoptotic effects of sorafenib, SC-1 and SC-43 in breast cancer cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-11-05.

The apoptotic ligand TRAIL is believed to have promise as a cancer gene therapy, yet many types of cancer, including gliomas, have exhibited resistance to TRAIL-induced apoptosis. Here, we show that therapeutic combination of the lipoxygenase inhibitor MK886 and TRAIL-secreting human mesenchymal stem cells (MSC-TRAIL) provide targeted and prolonged delivery of TRAIL both in vitro and in orthotopic mouse models of glioma. Treatment of either TRAIL-sensitive or TRAIL-resistant human glioma cells with MK886 and MSC-TRAIL resulted in significantly enhanced apoptosis compared with each agent alone. MK886 effectively increased the sensitivity to TRAIL-induced apoptosis via upregulation of the death receptor 5 and downregulation of the antiapoptotic protein survivin in human glioma cell lines and in primary glioma cells. This regulation was accompanied by a substantial increase in caspase activation after combined treatment. Furthermore, in vivo survival experiments and imaging analysis in orthotopic xenografted mice showed that MSC-based TRAIL gene delivery combined with MK886 into the tumors had greater therapeutic efficacy than single-agent treatment. Together, our findings indicate that MK886 combined with MSC-based TRAIL gene delivery may represent a novel strategy for improving the treatment of malignant gliomas. Cancer Res; 72(18); 4807–17. ©2012 AACR.

Survivin is an oncogene that functions in cancer cell cytoprotection and mitosis. Here we report that differential expression in cancer cells of a C-terminal splice variant of survivin, termed survivin-ΔEx3, is tightly associated with aggressive disease and markers of unfavorable prognosis. In contrast to other survivin variants, survivin-ΔEx3 localized exclusively to nuclei in tumor cells and was phosphorylated at multiple residues by the checkpoint kinase Chk2 during DNA damage. Mutagenesis of the Chk2 phosphorylation sites enhanced the stability of survivin-ΔEx3 in tumor cells, inhibited the expression of phosphorylated H2AX (γH2AX) in response to double-strand DNA breaks, and impaired growth after DNA damage. DNA damage induced Chk2 phosphorylation, stabilization of p53, induction of the cyclin-dependent kinase inhibitor p21, and homologous recombination–induced repair were not affected. In vivo, active Chk2 was detected at the earliest stages of the colorectal adenoma-to-carcinoma transition, persisted in advanced tumors, and correlated with increased survivin expression. Together, our findings suggest that Chk2-mediated phosphorylation of survivin-ΔEx3 contributes to a DNA damage–sensing checkpoint that may affect cancer cell sensitivity to genotoxic therapies. Cancer Res; 72(13); 3251–9. ©2012 AACR.

Cancer stem–like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC. Cancer Res; 72(11); 2844–54. ©2012 AACR.

Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is a bifunctional protein that has been implicated in both control of cell division and inhibition of apoptosis. Survivin has been shown to be involved in radiation resistance of various cancer types and its expression is increased by sublethal doses of irradiation in both differentiated and cancer stem cell (CSC) population. However, it is unknown whether suppression of survivin radiosensitizes the CSC population or diminishes its self renewal ability. Herein, we cloned the survivin dominant-negative mutant lacking a phosphorylation site (T34A) (Mesri et al., 2001 JCI) into the lentiviral LeGo vectors and assessed mammosphere formation and radiosensitization in MCF7, SUM149 and SUM159 cell lines grown under adherent or stem cell promoting conditions. We found an induction of survivin by western blotting in the dominant negative mutant (T34A)-transfected cell lines. Moreover, we observed a higher than two-fold increase in the Sub-G1 population as well as an increased Caspase 3 activity in the T34A-transfected SUM149 and SUM159 cells compared to control cells indicating an anti-apoptotic function of survivin. We also found that T34A-transfected cells showed a 1.5 to 2-fold decrease in the number of mammospheres compared to the control-transfected cells. Furthermore, T34A-transfected cells showed radiosensitization in adherent cells from SUM149 and SUM159 cells but no effect was observed in MCF7 cells. However, no radiosensitization was observed in stem cell promoting culture conditions with increasing doses of radiation in all tested cell lines. This indicates that the widely used standard clonogenic assays do not optimally select anti-CSC agents and that targeted therapies should be specifically tested for their activity against the CSC population. Further functional endpoint studies will be conducted to validate the in vitro findings. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-03.

Nasopharayngeal carcinoma (NPC) is an Epstein–Barr virus–associated malignancy most common in East Asia and Africa. Here we report frequent downregulation of the microRNA miR-218 in primary NPC tissues and cell lines where it plays a critical role in NPC progression. Suppression of miR-218 was associated with epigenetic silencing of SLIT2 and SLIT3, ligands of ROBO receptors that have been previously implicated in tumor angiogenesis. Exogenous expression of miR-218 caused significant toxicity in NPC cells in vitro and delayed tumor growth in vivo. We used an integrated trimodality approach to identify targets of miR-218 in NPC, cervical, and breast cell lines. Direct interaction between miR-218 and the 3′-untranslated regions (UTR) of mRNAs encoding ROBO1, survivin (BIRC5), and connexin43 (GJA1) was validated in a luciferase-based transcription reporter assay. Mechanistic investigations revealed a negative feedback loop wherein miR-218 regulates NPC cell migration via the SLIT-ROBO pathway. Pleotropic effects of miR-218 on NPC survival and migration were rescued by enforced expression of miR-218–resistant, engineered isoforms of survivin and ROBO1, respectively. In clinical specimens of NPC (n = 71), ROBO1 overexpression was significantly associated with worse overall (P = 0.04, HR = 2.4) and nodal relapse-free survival (P = 0.008, HR = 6.0). Our findings define an integrative tumor suppressor function for miR-218 in NPC and further suggest that restoring miR-218 expression in NPC might be useful for its clinical management. Cancer Res; 71(6); 2381–91. ©2011 AACR.