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Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

Common mechanisms for p53 loss in cancer include expression of MDM2 or the human papilloma virus (HPV)-encoded E6 protein which both mediate degradation of wild-type (WT) p53 (p53α). Here, we show that two alternatively-spliced, functional, truncated isoforms of p53 (p53β and p53γ, containing exons 1-9 of the p53 gene) can be markedly upregulated by pharmacologic or genetic inhibition of nonsense mediated decay (NMD), a regulator of aberrant mRNA stability. These isoforms lack the MDM2 binding domain and hence have reduced susceptibility to MDM2-mediated degradation. In MDM2-overexpressing cells bearing wildtypeTP53gene, NMD blockade increased p53β/γ expression and p53 pathway activation, enhanced radiosensitivity, and inhibited tumor growth. A similar pattern was observed in HPV+cancer cells and in cancer cells with p53 mutations downstream of exon 9. These results identify a novel therapeutic strategy for restoration of p53 function in tumors rendered p53 deficient through MDM2 overexpression, HPV infection, or certain p53 mutations.

Background: The serine/threonine kinase STK11/LKB1 is the second most commonly altered tumor suppressor in NSCLC. Non-functional mutations or loss of LKB1 expression occur more frequently in NSCLC than other alterations; however, there are currently no effective treatment strategies for this subset of tumors. KRAS-mutant LKB1 deficient NSCLC tumors often also have alterations in KEAP1 or NRF2 gene, which activate the KEAP1/NRF2 pathway known to be involved in antioxidant response. Inhibitors of ATM and ATR, two key proteins in the DNA damage response (DDR) pathway, are currently undergoing clinical testing but there are no validated biomarkers established for identifying which subgroups of patients are more likely to benefit from treatment. Here we have identified that alterations of LKB1, and the KEAP1/NRF2 pathway, are associated with enhanced response to ATM and ATR inhibitors (ATMi and ATRi) as well as other inhibitors of the DDR and may be useful biomarkers for predicting therapeutic response. Methods: To investigate the impact of LKB1 loss and KEAP1/NRF2 pathway activation on DDR and replication stress, we first tested replication fork protection in LKB1 deficient cells (KL). DNA fiber assay showed a defect in fork protection in KL cells compared with LKB1 wild type cells (K). Also, RPPA analysis revealed an activation of ATR/Chk1/Cdk1/CyclinB1 axis as well as Wee1 activation in cells harboring LKB1 and/or KEAP1 loss (KL/KLK/KK). Therefore, to evaluate response to DDR inhibitors (DDRi) we analyzed the in vitro activity of ATM, ATR, Wee1 and PARP inhibitors in NSCLC murine cell lines with or without knock out of LKB1 and/or KEAP1. In these cells, the loss of LKB1 and/or KEAP1 significantly sensitized to AZD0156 (ATMi), AZD6738 (ATRi) and AZD1775 (Wee1i) relative to cells with intact LKB1 and KEAP1. Next, we investigated whether the activity of ATR and ATMi in KL, KK or KLK tumor cells could be enhanced by the addition of a PARP inhibitor (olaparib). Although all NSCLC cells were resistant to the PARP inhibitor olaparib when used as a single agent, treatment of LKB1, KEAP1 or LKB1 plus KEAP1 deficient cells with the combination of olaparib plus ATM or ATR inhibitors significantly enhanced the antitumor cell activity of ATM or ATR inhibitors alone in vitro. We confirmed these data in an additional panel of LKB1 deficient NSCLC human cell lines treated with a broad spectrum of ATR and ATM inhibitors. In all human cell lines re-expression of LKB1 clearly reduced the sensitivity to ATR inhibition. LKB1 loss was also associated with sensitivity to PARP and ATM inhibitors, although these effects seemed to be less significant compared with ATR inhibitors. Interestingly, in vivo experiments performed in K, KL and KLK syngeneic models as well as PDX models showed greater response to ATRi and Wee1i monotherapy only in KLK but not in K or KL tumor models. Conclussions: Tumors with LKB1 deficiency or KEAP/NRF2 mutations are typically resistant to standard chemotherapy drugs and immunotherapy. Our data indicate that LKB1 and KEAP1/NRF2 loss significantly enhance the sensitivity to ATR, ATM and Wee1 inhibitors in vitro while only ATR and Wee1 inhibitor show significant tumor growth impairment in syngeneic and PDX KLK models. Thus, we have identified that NSCLC tumors bearing STK11 and KEAP1/NRF2 mutations are highly sensitive to DDR inhibitors and that genes may serve as biomarkers for selecting appropriate patients for treatment alone or in combination, such as PARPi, chemo or immunotherapy. Citation Format: Ana Galan-Cobo, Marlese Pisegna, Kavya Ramkumar, Alissa Poteete, Sungnam Cho, Fahao Zhang, You-Hong Fan, Qi Wang, Lixia Diao, Katharina Schlacher, Jing Wang, Lauren A Byers, John V. Heymcach. LKB1 deficiency and KEAP1/NRF2 pathway alterations as biomarkers of response for ATR and ATM inhibitors and other inhibitors of DNA damage response (DDR) in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A096. doi:10.1158/1535-7163.TARG-19-A096

The gold standard for determining the tumorigenic potential of human cancer cells is a xenotransplantation into immunodeficient mice. Higher tumorigenicity of cells is associated with earlier tumor onset. Here, we used xenotransplantation to assess the tumorigenic potential of human breast cancer cells following RNA interference-mediated inhibition of over 5000 genes. We identify 16 candidate tumor suppressors, one of which is the zinc-finger transcription factor SALL1. Analyzing this particular molecule in more detail, we show that inhibition of SALL1 correlates with reduced levels of CDH1, an important contributor to epithelial-to-mesenchymal transition. Furthermore, SALL1 expression led to an increased migration and more than twice as many cells expressing a cancer stem cell signature. Also, SALL1 expression correlates with the survival of breast cancer patients. These findings cast new light on a gene that has previously been described to be relevant during embryogenesis, but not carcinogenesis.

Oral premalignant lesion (OPL) expression of PD-L1 is associated with increased cancer risk. These data suggest that immune evasion is already present at the OPL stage and warrant an evaluation of immune modulatory therapies for cancer prevention. We conducted this preclinical study in a carcinogen-induced mouse model of oral cancer to evaluate the efficacy of multiple immunomodulatory strategies, given at a time point when only OPLs are present, to reduce the incidence of oral squamous cell carcinoma (OSCC). We treated C57BL/6 mice with the carcinogen 4-nitroquinoline 1-oxide (4-NQO) for 8 weeks in drinking water. Eight weeks after discontinuing 4-NQO treatment, a group of mice (N=6) was sacrificed for assessment of invasive and non-invasive tongue lesions. A second group of mice was treated with either anti-PD-1 (N=40), anti-CTLA-4 (N=20), anti-OX40 (N=20), anti-PD-1 + anti-CTLA-4 (N=20), anti-PD-1 + anti-OX40 (N=20) antibodies, or IgG (N=40) for a total of 3 doses every 3 days, initiating 8 weeks after the cessation of 4-NQO. Antibodies used in combination were given on the same day . Mice were sacrificed 56 days after the last dose of treatment. We assessed serial H&E-stained cross sections of the tongues harvested at that same time point for development of OPLs and cancer and measured the OSCC area of each tongue using aperio imagescope software. Mann-Whitney and Fisher's exact tests were used for statistical comparisons between groups. Eight weeks after 4NQO cessation, 100% of mice developed tongue hyperplasia or dysplasia and invasive lesions were not identified, indicating this to be an ideal time point to initiate treatment strategies addressing OPLs. Tongues from mice treated with anti-PD-1 antibody displayed a decrease in the mean OSCC area when compared with mice treated with IgG (mean 3.53 mm2 versus 6.62 mm2, respectively; p= 0.018). At this time point, invasive oral cancers had developed in 75% versus 50% of IgG and anti-PD-1 treated mice, respectively (p=0.03). There were also non-significant decreases in the mean OSCC area in tongues from mice treated with anti-CTLA-4 (mean = 5.07 mm2), anti-OX40 (mean = 3.79 mm2) and anti-PD-1 + anti-CTLA-4 (mean = 3.86 mm2) antibodies when compared to mice treated with IgG (mean = 6.62 mm2) control. In the group treated with anti-PD-1 + anti-OX40 combination therapy, there was an increase in the mean OSCC area when compared to anti-PD-1 monotherapy (mean 3.53 mm2 versus 9.03 mm2), respectively (p= 0.01). Short-term anti-PD-1 immune checkpoint inhibitor therapy in the context of OPLs led to a reduction in the incidence of oral cancer. When comparing the mean area of OSCC in each group, anti-PD-1 monotherapy was superior to IgG and all other treatment strategies. Paradoxically, combining anti-OX40 and anti-PD-1 antibodies created an antagonizing effect on the therapy and increased total tumor burden when compared with anti-PD-1 monotherapy. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Marlese A. Pisegna, Alissa Poteete, Fahao Zhang, John V. Heymach, William N. William. Anti-PD-1 monotherapy outperforms multiple immunotherapy combination strategies in an oral cancer prevention mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1268.

Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy. Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors. Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC. Citation Format: Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1719.

Acute myeloid leukemia (AML) 1 is often disrupted by chromosomal translocations generating oncogenic fusions in human leukemias. However, its role in epithelial cancers has not been extensively investigated. Herein, we show a marked accumulation of AML1 transcripts including a high frequency of a novel alternatively spliced AML1b transcript lacking exon 6 (AML1b(Del179-242)) in ovarian cancer patients. The increases in RNA transcripts for total wild-type AML1 and AML1b(Del179-242) are associated with poor patient outcomes. We have shown that although both wild-type AML1b and AML1b(Del179-242) are localized to nuclear speckles, AML1b(Del179-242) was observed to have dramatically reduced transactivation potential with the plasminogen activator inhibitor-1 promoters and behaved as a weak dominant negative of wild-type AML1b. Wild-type AML1b was found to inhibit the growth of immortalized ovarian epithelial cells (T29) decreasing colony-forming ability. Moreover, we have identified a novel function of AML1b where it inhibits ovarian cell migration. In contrast, AML1b(Del179-242) has lost the ability to inhibit both ovarian cell proliferation and migration indicating that the functional effects observed with wild-type AML1b are dependent on amino acids 179-242. Collectively, these studies suggest that deregulated alternative splicing of AML1b transcripts may potentially contribute to the pathophysiology of ovarian cancers.

We have previously reported that adenoviral vector-mediated interferon (IFN)-beta gene therapy inhibits orthotopic growth of human prostate cancer cells in nude mice. The purpose of this study was to determine efficacy and mechanisms of this therapy in immune-competent mice. TRAMP-C2Re3 mouse prostate cancer cells infected with 100 multiplicity of infection (MOI) of adenoviral vector encoding for mouse IFN-beta (AdmIFN-beta), but not AdE/1 (a control adenoviral vector), produced approximately 60 ng/10(5) cells/24 h of IFN-beta. The tumorigenicity of AdmIFN-beta-transduced cells was dramatically reduced in the prostates of C57BL/6 mice. A single intratumoral injection of 2 x 10(9) PFU (plaque-forming unit) of AdmIFN-beta inhibited tumor growth by 70% and prolonged survival of tumor-bearing mice. Intriguingly, this AdmIFN-beta therapy did not alter the growth of tumors in inducible nitric oxide synthase (iNOS)-null C57BL/6 mice. Immunohistochemical analysis revealed that treatment of tumors with AdmIFN-beta in wild-type C57BL/6 mice led to increased iNOS expression, decreased microvessel density, decreased cell proliferation, and increased apoptosis. Furthermore, quantitative reverse-transcriptional PCR analysis showed that AdmIFN-beta therapy, in C57BL/6 but not the iNOS-null counterparts, reduced levels of the mRNAs for angiopoietin, basic fibroblast growth factor, matrix metalloproteinase-9, transforming growth factor-beta1, vascular endothelial growth factor (VEGF)-A, and VEGF-B, as well as the antiapoptotic molecule endothelin-1. These data indicated that IFN-beta gene therapy could be effective alternative for the treatment of locally advanced prostate cancer and suggest an obligatory role of NO in IFN-beta antitumoral effects in vivo.

We investigated the role of transforming growth factor-β (TGF-β) signaling in the growth and metastasis of PC-3MM2 human prostate cancer cells. Highly metastatic PC-3MM2 human prostate cancer cells were engineered to constitutively overexpress a dominant-negative type II TGF-β receptor (DNR). Transfection of DNR had minimal direct effects on cell growth and attenuated TGF-β-induced cell growth inhibition and TGF-β1 production. There were no discernable differences in tumorigenicity (tumor incidence) among PC-3MM2 variants when the cells were implanted into the prostates of nude mice. Growth rate and metastatic incidence of DNR-engineered PC-3MM2 cells, however, were significantly reduced. Most cells in the control tumors were positively stained by an antibody to proliferation cell nuclear antigen and very few cells were stained by terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL). In sharp contrast, tumors formed by PC-3MM2-DNR cells contained fewer proliferation cell nuclear antigen–positive cells and many more TUNEL-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3MM2-DNR tumors. Expression of interleukin-8 (IL-8) in tumors formed by PC-3MM2 cells was significantly reduced as revealed by both Northern blotting and ELISA. Finally, transfection of antisense IL-8 cDNA significantly reduced IL-8 production by PC-3MM2 cells and antisense IL-8-transfected PC-3MM2 cells grew slower in comparison with parental and control vector-transfected cells. Taken together, our data suggest that TGF-β signaling, by regulating IL-8 expression in tumor cells and hence tumor angiogenesis, is critical for progressive growth of PC-3MM2 cells in the prostate of nude mice.

Introduction: Immunotherapy with single agent anti-PD-1 antibody confers modest overall survival benefits in patients with recurrent/metastatic HNSCCs. Combined immune checkpoint targeting has shown promising results for melanomas and other solid tumors. Thus, evaluation of combination immunotherapy in animal models specific to HNSCCs could inform prioritization and design of human clinical trials for this disease. We conducted this pre-clinical study to test the hypothesis that combining anti-PD-1 with either anti-CTLA-4 or co-stimulatory immune checkpoint agonist antibodies would be more effective than anti-PD-1 monotherapy in a mouse model of oral carcinoma. We also aimed at identifying the combination regimen with highest anti-tumor activity. Methods: C57BL/6 mice were subcutaneously grafted with 2x106 mouse oral cancer 1 (MOC1) cells derived from a carcinogen-induced tumor molecularly similar to human HNSCC. After 25 days, mice were randomized into one of 12 treatment groups (IgG, or antibodies targeting PD-1, CTLA-4, CD40, OX-40, GITR, 4-1BB, PD-1+CTLA-4, PD1+CD40, PD-1+OX-40, PD-1+GITR, or PD-1+4-1BB), N=8-9 mice/group. Antibodies were administered every 4 days for 3 doses. Tumors were measured twice per week. The primary endpoint was overall survival. Mice were sacrificed when tumor diameter was >2 cm or tumor ulceration was >5 mm. Survival curves were computed using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. Results: In analogy to human clinical trials, anti-PD-1 therapy led to a modest improvement in survival compared to IgG [HR=0.41; 95%CI 0.06-0.53; P=0.015], indicating that this model recapitulates, in part, the effects of immunotherapy in HNSCC patients; none of the animals survived long term. Monotherapy with anti-CTLA-4, anti-CD40, anti-OX-40, or anti-4-1BB also improved survival compared to IgG (P=0.0001, 0.003, 0.02, and 0.003, respectively), but were not more effective than anti-PD-1 alone (P=0.09, 0.12, 0.72, and 0.77, respectively). Combination therapy did not prolong survival compared to anti-PD-1 alone, with the exception of anti-PD-1+ anti-CTLA4 [HR=0.36; 95% CI 0.07-0.6; P=0.016], and anti-PD-1+ anti-CD40 [HR=0.125, 95% CI 0.02-0.24; P=0.0003]. Notably, 50% of mice receiving anti-PD-1+ anti-CD40 antibodies had complete tumor eradication and are alive and disease-free 120 days after tumor grafting. Conclusions: Single agent immunotherapy targeting PD-1, CTLA-4, CD40, OX-40 and 4-1BB modestly improved survival in a mouse model of oral cancer. Combination with anti-PD-1+ anti-CTLA4 and anti-PD-1+ anti-CD40 antibodies outperformed anti-PD-1 monotherapy. Complete responses were exclusively seen with PD-1+CD40 dual targeting. This regimen should be prioritized for evaluation in HNSCC clinical trials. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Alissa R. Poteete, Marlese A. Pisegna, Uma Giri, Fahao Zhang, Patrick Hwu, John V. Heymach, William N. William. Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5648. doi:10.1158/1538-7445.AM2017-5648

The purpose of this study was to determine the effects of interferon-beta (IFN-beta) gene transfer on the growth of PC3MM2 human prostate cancer cells in nude mice. Intralesional delivery of an adenoviral vector encoding murine IFN-beta (AdIFN-beta), but not a vector encoding bacterial beta-galactosidase (AdLacZ), suppressed PC3MM2 tumors in a dose-dependent manner. At the highest dose (2x10(9) plaque-forming units, PFU), a single injection of AdIFN-beta (but not AdLacZ) suppressed orthotopic PC3MM2 tumors and development of metastasis by 80%, and eradicated the tumors in 20% of mice. Immunohistochemical staining showed that AdIFN-beta-treated tumors contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did the control tumors. Compared with controls, tumors injected with AdIFN-beta expressed higher levels of IFN-beta and inducible nitric oxide synthase (iNOS) and lower levels of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). In vitro analysis indicated that expression of bFGF and TGF-beta1 in PC3MM2 cells could be suppressed by the nitric oxide donor sodium nitroprusside. These data suggest that intratumoral delivery of the IFN-beta gene with adenoviral vectors could be an effective therapy for prostate cancer and that tumor suppression by AdIFN-beta correlated with up-regulation of iNOS and down-regulation of angiogenesis.

Introduction Tumor cells growing in the brain are resistant to chemotherapy. We have recently demonstrated that the activation of the endothelin receptors A and B axis is involved in the induction of resistance to chemotherapy by primary brain tumor cells and by brain metastases. Treatment with a dual endothelin receptor antagonist in combination with chemotherapy produced significant therapy of orthotopically growing glioblastoma cells as well as breast cancer and lung cancer brain metastases. Since the milieu of tumors in the brain is hypoxic (0.5% - 1% O2), we investigated whether the chemo-resistance of the tumor cells is linked to hypoxia mediated activation of the endothelin receptor axis. Materials and Methods Human glioma (LN229), breast cancer (MDA231), and lung adenocarcinoma (PC14) cell lines were cultured in DMEM supplemented with 5% FBS. The endothelin receptor A (ETAR) and/or B (ETBR) of cancer cells were knocked down by shRNA. ETAR and ETBR were activated by incubating the tumor cells with exogenous endothelin-1 for 15 minutes or blocked by treating the parental or engineered cells with ETAR-specific antagonist, BQ123 (100nM), and/or ETBR-specific antagonist, BQ788 (100nM), for 2 hours prior to the addition of paclitaxel (5ng/ml), temozolomide (100μg/ml) or cisplatinum (5μg/ml). The cultures were placed into incubators with an atmosphere of 20%, 6% or 1% oxygen for 48 hours (MDA231 cells) or 72 hours (LN229 and PC14 cells). Tumor cells were then plated into 96-well plates to determine chemosensitivity by the MTT assay or into 6-well plates for western blots, or into chamber slides for immunohistochemical analyses. Expression of survival-related proteins such as pETAR, pETBR, pAkt, pMAPK, pNFκB, GSTA5, TWIST1 or Bcl2L1, were then determined. Results Stimulation of parental MDA-231, PC-14, or LN229 cells with exogenous ET-1 induced significant resistance against all tested chemotherapeutic agents. The resistance was abolished only when both the ETAR and ETBR were antagonized by a combination of BQ123 and BQ788. Parental cells cultured under hypoxia were also significantly resistant to chemotherapy and treatment of these cells with BQ123 and BQ788 reversed this resistance. The effects of exogenous ET-1 on the induction of chemo-resistance or BQ123 and BQ788 on restoration of chemo-sensitivity were not found in any cancer cells devoid of ETAR and ETBR. In all cases, the chemo-resistance of tumor cells was associated with increased level of expression of proteins related to cell survival. Conclusion These data suggest that the endothelin receptor axis plays a role in hypoxia induced resistance of cancer cells to chemotherapy. Additional studies are warranted to determine the functional changes of the endothelin receptor axis in hypoxia. Citation Format: Hyun Kyung Yu, Ho Jeong Lee, Fahao Zhang, Qiuyu Wu, Isaiah J. Fidler, Sun Jin Kim. Hypoxia induced resistance to chemotherapy is regulated by the endothelin receptor axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2943.

PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.

During lung development, cells proliferate for a defined length of time before they begin to differentiate. Factors that control this proliferative process and how this growth process is related to lung cancer are currently unknown. Here, we found that the WD40-containing protein (p44/wdr77) was expressed in growing epithelial cells at the early stages of lung development. In contrast, p44/wdr77 expression was diminished in fully differentiated epithelial cells in the adult lung. Loss of p44/wdr77 gene expression led to cell growth arrest and differentiation. Re-expression of p44/wdr77 caused terminally differentiated cells to re-enter the cell cycle. Our findings suggest that p44/wdr77 is essential and sufficient for proliferation of lung epithelial cells. P44/Wdr77 was re-expressed in lung cancer, and silencing p44/wdr77 expression strongly inhibited growth of lung adenocarcinoma cells in tissue culture and abolished growth of lung adenocarcinoma tumor xenografts in mice. The growth arrest induced by loss of p44/wdr77 expression was partially through the p21-Rb signaling. Our results suggest that p44/wdr77 controls cellular proliferation during lung development, and this growth process is reactivated during lung tumorigenesis.

Potential treatments for ovarian cancers that have become resistant to standard chemotherapies include modulators of tumor cell survival, such as endothelin receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1, growing orthotopically in nude mice. Mice with established disease were treated with vehicle (control), paclitaxel (weekly, intraperitoneal injections), macitentan (daily oral administrations), or a combination of paclitaxel and macitentan. Treatment with paclitaxel decreased tumor weight and volume of ascites. Combination therapy with macitentan and paclitaxel reduced tumor incidence and further reduced tumor weight and volume of ascites when compared with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but alone had no effect on tumor incidence or ascites. Immunohistochemical analyses revealed that treatment with macitentan and macitentan plus paclitaxel inhibited the phosphorylation of ETRs and suppressed the survival pathways of tumor cells by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan necessary for inhibition of phosphorylation correlated with the dose required to increase antitumor efficacy of paclitaxel. Treatment with macitentan enhanced the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis in tumor cells and tumor-associated endothelial cells. Collectively, these results show that administration of macitentan in combination with paclitaxel prevents the progression of ovarian cancer in the peritoneal cavity of nude mice in part by inhibiting survival pathways of both tumor cells and tumor-associated endothelial cells.

Murine melanomas produce site-specific experimental brain metastases that reflect clinical reality. When injected into the internal carotid artery of mice, K-1735 melanoma cells produce metastatic lesions only in the brain parenchyma, whereas B16 melanoma cells and the somatic hybrid cells of B16 × K-1735 melanoma cells produce metastatic lesions only in the leptomeninges and ventricles. In the present study, we identified transforming growth factor-β2 (TGF-β2), an isoform of the TGF-β family, as a molecular determinant of melanoma cell growth in the brain parenchyma. We found that the TGF-β2 mRNA was highly expressed by the K-1735 cells, whereas the B16 cells or any B16 × K-1735 somatic cell-cell fusion hybrids have low expression. Transfection of the TGF-β2 gene into B16 cells resulted in the production of microscopic metastatic lesions in the brain parenchyma, without a decrease in metastasis to the leptomeninges or ventricles. TGF-β2 knockdown in the K-1735 melanoma cells significantly reduced metastasis to the brain parenchyma but did not induce metastasis to the leptomeninges or ventricles. These data show that TGF-β2 expression by murine melanoma cells is necessary for the establishment and growth of metastases in the brain parenchyma. [Cancer Res 2009;69(3):828–35]

GN963 is a tyrosine kinase inhibitor with activity against platelet-derived growth factor receptor (PDGFR) and Src kinases. We determined whether oral administration of GN963, alone or in combination with gemcitabine produces therapy against L3.6pl human pancreatic cancer cells growing orthotopically in nude mice. The optimal biological dosage of oral GN963 was determined to be 100 mg/kg every 48 h. Seven days after injection of L3.6pl cells into the pancreas of nude mice, mice (n=10) were treated with vehicle (control), thrice-weekly oral GN963 (100 mg/kg), twice-weekly intraperitoneal gemcitabine (100 mg/kg), or GN963 plus gemcitabine. Treatment with gemcitabine did not significantly differ from control. In contrast, treatment with GN963 (100 mg/kg) or GN963 plus gemcitabine produced a 52% and 81% decrease in tumor volume, respectively. GN963 plus gemcitabine completely inhibited the incidence of liver metastasis. Administration of GN963 inhibited PDGFR phosphorylation in both tumor and tumor-associated endothelial cells, decreased Src and Akt kinase activity in tumor cells, decreased microvessel density, and decreased tumor cell proliferation, while increasing apoptosis of tumor and tumor-associated endothelial cells. Collectively, these data indicate that targeting PDGFR, Src, and Akt on tumor and tumor-associated endothelial cells may be an effective therapy for human pancreatic carcinoma.

Androgen receptor (AR) is a ligand-activated transcription factor that mediates the action of androgens and is essential for the growth, function, and cell differentiation of the prostate gland. Here, we demonstrated that the prostate apoptosis response factor-4 (par-4) functions as a novel AR coactivator. Par-4 physically interacted with the DNA-binding domain of AR, enhanced AR interaction with DNA, and increased AR-dependent transcription. Par-4 enhanced the c-FLIP promoter activity and was recruited on to the c-FLIP gene in the presence of androgens, and the dominant-negative par-4 decreased c-FLIP expression. These results suggest that, in addition to its proapoptotic function, par-4 acts as a novel transcription cofactor for AR to target c-FLIP gene expression. In addition, we demonstrated that loss of c-FLIP expression was essential for castration-induced apoptosis in the prostate gland and that enhanced c-FLIP expression was associated with prostate cancer progression to the androgen-resistant stage. Our data shed light on a transcription-mediated mechanism for the effects of the AR pathway on cell survival and apoptosis.

Interleukin (IL)-8 and transforming growth factor (TGF)-beta1 are proangiogenic factors overexpressed in advanced human melanoma. We investigated the effects of TGF-beta1 on IL-8 expression in the well-characterized A375 human melanoma system. We demonstrated by enzyme-linked immunoassay and Northern blot analysis that TGF-beta1 selectively induced IL-8 expression, at both protein and mRNA levels, in highly metastatic A375SM cells but not cells of their poorly metastatic parental line A375P. Transient transfection with luciferase reporter gene constructs revealed that TGF-beta1 activated IL-8 promoter activity in A375SM cells but not A375P cells. Studies with progressive 5' deletion constructs and site-specific mutations demonstrated that a construct containing -133 to +44 of the 5'-flanking sequence was necessary and sufficient for maximal TGF-beta1-induced transcription response and that TGF-beta1-induced activation of IL-8 promoter depended on AP-1 (-126 to -120 bp), NF-kappaB (-94 to -71 bp), and C/EBP-like factor NF-IL6 (-94 to -81 bp) in this region. Interestingly, both A375P and A375SM cells expressed type I and type II TGF-beta receptors and TGF-beta1 induced the nuclear translocation of Smad3 protein in both A375P and A375SM cells. Moreover, both A375P and A375SM cells were susceptible to TGF-beta1-induced growth inhibition. Our data thus demonstrated that TGF-beta1 selectively induced IL-8 expression in highly metastatic A375SM melanoma cells. This TGF-beta1-induced IL-8 expression could be an amplification cascade responsible for overexpression of IL-8 in human melanoma and one of potential mechanisms by which TGF-beta1 promotes angiogenesis, growth, and metastasis of human melanoma.

Introduction: In 2009, ovarian cancer was the leading cause of death from gynecologic cancer in the United States. Efforts to develop tools for early detection and therapeutic regimens to overcome drug resistance of the ovarian cancer have not made a significant achievement to prolong the median survial of the patients. Potential treatments for ovarian cancers that have become resistant to standard chemotherapies include modulators of tumor cell survival, such as endothelin receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1, growing orthotopically in nude mice. Materials and Methods: Mice were injected with one million cells of SKOV3ip1 or IGROV1. Ten days later, mice with established disease were randomized into vehicle (control), paclitaxel (weekly, intraperitoneal injections), macitentan (daily oral administrations) or a combination of paclitaxel and macitentan treatment groups. After 4 weeks of treatment, mice were necropsied and tumor incidence, tumor weight, and incidence of ascites were recorded. Tumor tissues were processed for immunohistochemical analyses. Results: Treatment with paclitaxel decreased tumor weight and volume of ascites. Combination therapy with macitentan and paclitaxel reduced tumor incidence and further reduced tumor weight and volume of ascites when compared with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but alone had no effect on tumor incidence or ascites. Immunohistochemical analyses revealed that treatment with macitentan and macitentan plus paclitaxel inhibited the phosphorylation of ETRs and suppressed the survival pathways of tumor cells by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan necessary for inhibition of phosphorylation correlated with the dose required to increase antitumor efficacy of paclitaxel. Conclusion: Treatment with macitentan enhanced the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis in tumor cells and tumor-associated endothelial cells. Collectively, these results show that administration of macitentan in combination with paclitaxel prevents the progression of ovarian cancer in the peritoneal cavity of nude mice in part by inhibiting survival pathways of both tumor cells and tumor-associated endothelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2011-601