Search

Systemic metastasis is the major cause of death from melanoma, the most lethal form of skin cancer. Although most patients with melanoma exhibit a substantial gap between onset of primary and metastatic tumors, signaling mechanisms implicated in the period of metastatic latency remain unclear. We hypothesized that melanoma circulating tumor cells (CTC) home to and reside in the bone marrow during the asymptomatic phase of disease progression. Using a strategy to deplete normal cell lineages (Lin−), we isolated CTC-enriched cell populations from the blood of patients with metastatic melanoma, verified by the presence of putative CTCs characterized by melanoma-specific biomarkers and upregulated gene transcripts involved in cell survival and prodevelopment functions. Implantation of Lin− population in NSG mice (CTC-derived xenografts, i.e., CDX), and subsequent transcriptomic analysis of ex vivo bone marrow–resident tumor cells (BMRTC) versus CTC identified protein ubiquitination as a significant regulatory pathway of BMRTC signaling. Selective inhibition of USP7, a key deubiquinating enzyme, arrested BMRTCs in bone marrow locales and decreased systemic micrometastasis. This study provides first-time evidence that the asymptomatic progression of metastatic melanoma can be recapitulated in vivo using patient-isolated CTCs. Furthermore, these results suggest that USP7 inhibitors warrant further investigation as a strategy to prevent progression to overt clinical metastasis. Significance: These findings provide insights into mechanism of melanoma recurrence and propose a novel approach to inhibit systematic metastatic disease by targeting bone marrow-resident tumor cells through pharmacological inhibition of USP7. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5349/F1.large.jpg. Cancer Res; 78(18); 5349–62. ©2018 AACR.

Sorafenib is the only first line and FDA-approved drug available for the treatment of advanced hepatocellular carcinoma (HCC). Despite an increased overall patient survival of about 3-5 months with sorafenib, majority of the HCC patients acquire sorafenib-resistance, thus making sorafenib treatment ineffective in long-term. Therefore, there is an urgent need to explore the mechanisms underlying sorafenib-resistance to develop better treatment strategies to combat this deadly disease. To achieve this goal, we first established sorafenib-resistant cells by repeated treatment of different HCC cell lines (Huh7, Hep3B and HepG2) with increasing concentrations of sorafenib up to 6µM. MTT assays confirmed that all three cell types treated with sorafenib achieved resistance and were termed soraR to distinguish them from their sorafenib-sensitive (soraS) counterparts. Further characterization of the soraR cells showed increased expression of mesenchymal markers including Slug, Snail, Zeb2, vimentin, and reduced expression of epithelial marker (E-cadherin), indicative of Epithelial-Mesenchymal Transition (EMT). Moreover soraR cells were found to have high migratory potential in wound healing assay and higher sphere-forming capability compared to the soraS cells. To find out the signaling pathways which could be responsible for promoting sorafenib-resistance, RT2-profiler PCR Array analysis for cytokine and chemokines (PAHS-150Z from Qiagen) were performed, which revealed an increase in the expression of BMP6 and BMP7 mRNA levels in the soraR cells. The results were validated by qPCR. In addition, western blot analyses showed an increase in pSmad1, 5, 9 levels in the soraR cells, suggesting a potential activation of BMP6, 7 axis in sorafenib-resistance. Effect of inhibition of BMP6 and BMP7 signaling in re-sensitizing the SoraR cells towards sorafenib was determined next. Flow cytometric analysis of Hep3B-SoraR cells treated with DMH1, a small molecule inhibitor of BMP receptors in combination with sorafenib showed increased cell death which was accompanied with increased expression of cleaved PARP and cleaved caspase 3 in western blot. BMP6 and BMP7 inhibition also reduced migration capacity of soraR cell types as examined by wound healing assay. The results were further confirmed by treating soraR cells with recombinant BMP6 and BMP7 and examining their effect on migration and sorafenib-mediated apoptosis. Interestingly, exogenous BMP6 and BMP7 treatment was found to enhance migration capacity and rescue the soraS cells from sorafenib-mediated cell death. These findings indicated an important role of BMP6 and BMP7 pathways in mediating sorafenib-resistance in HCC and suggested that pharmacological targeting of BMP6 and BMP7 signaling could be an effective strategy to ameliorate sorafenib-resistance in advanced HCC. Citation Format: Kanchan Vishnoi, Rong Ke, Randhir Kumar, Navin Viswakarma, Ajay Rana, Basabi Rana. Elucidation of the molecular mechanisms mediating sorafenib-resistance in Hepatocellular carcinoma [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 2614.

Hepatocellular carcinoma (HCC) is one of the most common gastrointestinal (GI) malignancies and a major cause of death worldwide, with limited therapeutic options. Evidence suggest that Mixed Lineage Kinase 3 (MLK3), a member of MAPK pathway, is a key regulator of tumorigenesis and metastasis in different cancer-types. Previous studies from our laboratory indicated that, knocking out MLK3 in mice alleviates hepatocarcinogenesis when treated with Diethylnitrosamine (DEN) in combination with phenobarbital (PB). While these suggested an involvement of MLK3 in HCC progression, the detailed mechanism by which this is accomplished is still unclear. To address the mechanism of MLK3-induced HCC, liver cancer cells stably overexpressing MLK3 wildtype (WT) or kinase dead (KA) in a Doxycycline (Dox)-inducible manner, were established. Our RT2 Profiler PCR array analysis with a cytokine and chemokine array (PAHS-150Z from QIAGEN) showed that MLK3-WT overexpression can significantly upregulate mRNA levels of inflammatory cytokines, such as IL11, BMP6, TNFα and IL6, which were absent with MLK3-KA overexpression. Q-PCR and ELISA analysis confirmed the increased IL11 mRNA levels and IL11 release following MLK3 overexpression. Interestingly, STAT3, the downstream signal transducer of Janus kinases (JAKs), which are activated by cytokine signals was also activated by MLK3 overexpression, suggesting that MLK3 may have a novel crosstalk with JAK-STAT3 signaling pathway. To explore this possibility, cells were treated with inhibitors of JAKs (AZD1480 and Ruxolitinib) in the presence of Dox (to induce MLK3), which significantly decreased STAT3 activation as well as IL11 release, suggesting the involvement of JAKs in MLK3-induced IL11 signaling. Furthermore, we also demonstrated a significant reduction of IL11 release following treatment with the JNK inhibitor, SP600125. To elucidate the detailed mecahnism and identify potential transcription factors that mediate the effect of MLK3 overexpression on IL11 promoter activity, deletion mutants of pGL4.16 IL11-luciferase reporter (-877/+169), (-249/+169), (-153/+169) and (-78/+169) were generated. Luciferase assays confirmed that MLK3-WT overexpression indeed increased IL11 promoter reporter activity, which required the fragment from -153 to -78. More studies are currently undergoing with site-directed mutagenesis to explore the detailed mechanism. In addition, our studies demonstrated an induction of epithelial to mesenchymal transition (EMT) during later stages of Dox treatment, suggesting that MLK3-WT overexpression may regulate EMT, cell migration and invasion. These results indicated that both JAKs, and MLK3/JNK signaling converge to mediate IL11 release in HCC, but the mechanism of crosstalk between MLK3 and JAK-STAT3 signaling pathway is still unclear. Taken together, our current studies indicate that overexpression of MLK3 may not only activate JNK/c-Jun pathway, but also regulate JAK-STAT3 signaling pathway to induce IL11 release and downstream signaling in hepatocellular carcinoma. Citation Format: Rong Ke, Navin Viswakarma, Kanchan Vishnoi, Sandeep Kumar, Ajay Rana, Basabi Rana. Elucidation of MLK3-induced signaling pathways in hepatocellular carcinoma [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 108.

Despite widespread knowledge that bone marrow-resident breast cancer cells (BM-R BCs) affect disease progression and ultimately cause death, mechanisms regulating patient-derived BM-R BC quiescence remain poorly defined. This is caused by lack of biomarkers that can isolate quiescent but viable BM-R BCs devoid of cytokeratin and/or other epithelial markers. We developed and validated an experimental strategy to capture, isolate and characterize identify and isolate solitary BC cells from blood and BM of xenograft mice using a combination of human and BC-specific markers. These procedures allowed us to interrogate not only cytokeratin-positive but also unidentified cytokeratin-negative BC cell subsets. We hypothesized that subsets of patient-isolated circulating tumor cells (CTCs) will re-colonize various organs; and that underlying CTC signaling mechanisms will reflect respective transcriptomic signatures upon xeno-transplantation. Accordingly, we established an in vivo model of “latent” metastasis in CTC-derived xenografts (CDXs) by three sequential steps: a) isolation of CTC-enriched populations from metastatic BC patients, followed by their implantation in NSG mice; b) in vivo depletion of “non-tumor” human cells over an 8-month period; and c) capture and characterization of viable BC cells from blood and BM of CDX mice. These steps were implemented on patient samples from all primary BC subtypes along with validation of ex vivo CTC and BM-R BC gene signatures by whole genome transcriptomic arrays. First, three-way transcriptomic comparison between ex vivo BM-R BCs and CTCs vs de novo CTCs (obtained from GSE99394) revealed that ex vivo BMRBCs and CTCs clustered together and were largely similar (279 differentially regulated genes), implying that either the majority of ex vivo CTCs were shed from BM or that CTC transcriptomic signatures shed from other organs were similar to BM-shed CTCs. Second, comparative analyses of ex vivo BM-R BC vs de novo CTC gene signatures identified heightened mTORC2 vis-à-vis attenuated mTORC1 signaling as the most significant parameter of human BM-R BCs. Third, augmented levels of mTORC2-downstream targets were detected in quiescent (Ki67-/RBL2+) cells of paired metastatic vs primary BC tissues. Fourth, IHC analyses of CTC xenograft tissues revealed solitary BM and tissue-resident BC cells possessing high mTORC2. Lastly, shRNA knockdown of Rictor - the essential component of the mTORC2 complex - stimulated cell cycle progression and proliferation in vitro while depleting the BM-R BC population in vivo. Collectively, these findings suggest that the balance between mTORC2 vs mTORC1 interplays regulate the primordial stages of CTC quiescence. Deciphering mTORC2 signaling in CTC quiescence will proffer novel biomarkers for quiescent CTC detection and innovative therapeutic interventions in BC patients with undetectable metastasis. Citation Format: Debasish Boral, Monika Vishnoi, Haowen Liu, DARIO MARCHETTI. Mechanisms driving clinical bone marrow-resident cancer cell quiescence [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 3387.

Sorafenib is the only FDA-approved drug currently available for the treatment of advanced hepatocellular carcinoma (HCC). However, development of sorafenib-resistance in patients poses a major challenge to make this treatment successful in long-term. There is an urgent need to explore the mechanisms underlying sorafenib-resistance and to develop strategies towards overcoming sorafenib-resistance. Aberrant activation of Wnt/β-catenin signaling in HCC is suggested to maintain tumor initiating cells, drug-resistance, tumor progression, and metastasis. Combining β-catenin inhibitors with sorafenib might thus be an effective therapeutic strategy to target drug-resistant HCC. To explore this, we used wnt/β-catenin pathway inhibitor XAV-939 in combination with sorafenib to study their effect on the apoptotic potential of HCC cell lines Huh7, Hep3B and HepG2. XAV-939 was demonstrated to effectively reduce the expression of β-catenin in wild-type expressing Huh7 and Hep3B cells. Flow cytometric analysis of these cells pre-treated with XAV-939 followed by sorafenib treatment showed a significant increase in apoptosis in a time dependent manner. Furthermore, these cells revealed increased mitochondrial depolarization which was accompanied by a parallel increase in cytosolic cytochrome c release and downregulation of pro-survival protein Mcl-1. These effects of XAV-939 were further validated following knockdown of endogenous β-catenin, which showed a potentiation of sorafenib effects with β-catenin knockdown. Further, to understand the mechanism associated with sorafenib-resistance, we developed sorafenib-resistant HCC cells by repeated exposure to increasing concentration of sorafenib. To our surprise the combination of sorafenib and XAV-939 was ineffective in increasing the apoptotic potential of these resistant cells, suggesting a differential role of wnt/β-catenin axis in sorafenib-resistance. Taken together, these results suggested that combination of wnt/β-catenin pathway inhibitor and sorafenib could be effective in sensitizing HCC cells to apoptosis initially, but might be ineffective once these cells acquire sorafenib-resistance. Citation Format: Kanchan Vishnoi, Rong Ke, Navin Vishwakarma, Ajay Rana, Basabi Rana. XAV-939, a Wnt/beta-catenin pathway inhibitor, sensitizes liver cancer cells to sorafenib: Implications in sorafenib resistance [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 2338.

Metastatic “seeding” is an early event initiated when cancer cells are shed from the primary tumor, enter into the blood stream as circulating tumor cells (CTCs), and travel to distant organs where they can remain dormant as disseminated cancer cells (DTCs) for indefinite lengths of time. The organ serving as the foremost reservoir for DTCs is the bone marrow (BM). Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. This is caused by the lack of biomarkers that can identify and isolate viable but dormant BMRCs not expressing neoplastic and/or proliferation markers. To overcome these hurdles, we developed a patient-derived CTC model of clinical dormancy. We hypothesized that a significant distinction exists between signaling pathways of BM-homing vs metastasis-competent CTCs upon transplantation in xenografts. The model was established by three sequential steps: (a) isolation of CTC-enriched cell populations from the peripheral blood of patients with metastatic breast cancer, followed by CTC implantation in NOD-SCID-gamma mice; (b) in vivo depletion of “normal” human cell lineages over 4-8 months (before onset of overt metastasis), and (c) capture and characterization of viable human CTCs from blood and BM of mice via multi-parametric flow cytometry. This strategy was implemented on a set of breast cancer patients stratified according to their primary tumor subtype (ER+/PR+, HER2+, and ER-/PR-/HER2-), and gene-signatures of putative ex vivo CTCs and BMRCs were obtained by whole genome transcriptomic arrays. First, comparative analyses of ex vivo vs de novo CTC-gene signatures identified increased mTORC2 along with decreased mTORC1 signaling activity as the most significant characteristic of human BM-resident CTCs. mTORC2/mTORC1 represent the two complementary arms of mTOR signaling - a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy. Second, heightened mTORC2 downstream targets augmented quiescent CTC populations (Ki67-/RBL2+ cells) in paired metastatic vs primary breast cancer tissues. Third, IHC analyses of breast cancer CTC xenograft tissues showed that solitary BM and tissue-resident breast cancer CTCs had high mTORC2 activity. Finally, shRNA knockdown of Rictor-the essential component of mTORC2 actions, increased Ki67/PCNA expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling pathways regulate CTC-associated mitotic and/or dormancy characteristics. Further elucidation of mTOR-mediated CTC dormancy will provide novel strategies for therapeutic interventions in breast cancer patients having metastatic “seeding” but yet to develop overt metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio T. Scamardo, David S. Hong, Dario Marchetti. Targeting bone marrow-resident dormant CTCs to overcome metastasis [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 3264.

Recurrence is the major cause of melanoma death due to cell dissemination from primary/metastatic tumor (CTCs). During asymptomatic periods, these cells reside in bone marrow (bone-marrow resident tumor [BMRT cells]) and remain quiescent. We hypothesized that disseminated tumor cells survive in bone marrow (BM) during these periods and evolve to metastatic potency during disease progression. First, we isolated CTC-enriched, Lin-neg population from clinically advanced melanoma patients and performed biomarker expression and mutational profiling to confirm presence of putative CTCs. Lin-neg CTC population contained unique transcriptomics signature with elevated melanoma markers expression (BAGE, MAGEA1, B4GALNT1, S100A3). Further, downstream IPA analysis demonstrated that an upregulation of transcripts for genes involved in cell survival and pro-development functions with concomitant decrease in cell proliferative and inflammation properties. Second, we implanted Lin-neg population in murine xenograft models and isolated HLA+/ Melan-A+ cells population from blood and BM at endpoint. Expression of human (HLA-ABC) and melanoma markers (Melan-A, S100, NG2, CD146) on BMRTCs and CTCs was confirmed by employing: (a) immunofluorescence staining, (b) single-cell DEPArrayTM and CellSearchTM capture, (c) genomic profiling, and (d) organ-site colonization of CTCs with concomitant to BMRTCs in BM. Third, we discovered distinct transcriptomic signature of BMRTCs vs CTCs by differential gene expression profiling. Subsequent pathway analysis showed top five altered canonical pathways in BMRTCs: protein ubiquitination pathway, EIF2, actin cytoskeleton, systemic lupus erythematosus and hypoxia signaling. Of note, a strikingly elevated expression of PTEN in BMRTCs was detected. Fourth, because PTEN binds to USP7, a key component of protein ubiquitination pathway, we evaluated USP7/PTEN axis in CTC-driven BMRTCs modulated metastatic competency. The use of two clinically approved USP7 inhibitors to study their effects on the proliferative capacity of BMRTCs led to a significant reduction of CTCs at metastatic sites. This study provides critical insights to identify biomarkers of melanoma recurrence during the asymptomatic periods of the disease, fostering application of USP7 inhibitors for innovative melanoma therapies for patients with metastasis undetectable disease and/or yet to develop metastasis. Citation Format: Monika Vishnoi, Debasish Boral, Haowen N. Liu, Marc L. Sprouse, Wei Yin, Michael A. Davies, Isabella C. Glitza Oliva, Dario Marchetti. Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells [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 3601.

Resistance towards standard therapeutic regimens and evasion of apoptosis are some of the hallmarks of advanced forms of cancer, which include Sorafenib-resistance in hepatocellular carcinoma (HCC), castration-resistance in prostate cancer. Development of effective therapeutic strategies that can target these resistant forms is critically needed. In an effort to understand the molecular mechanism mediating resistance in cancer, in previous studies utilizing Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells we showed that combination of TRAIL along with the PPARγ ligand Troglitazone (TZD) can sensitize them towards apoptosis. Using more molecular approaches these studies identified the serine/threonine protein kinase, AMP-activated protein kinase (AMPK) as a mediator of TRAIL-TZD-induced apoptosis. Since AMPK seemed to be a major mediator of this apoptosis, in the current studies we utilized the natural compound Berberine (BBR), a known activator of AMPK in combination with TRAIL. These demonstrated a significant reduction of cell viability (MTT assay) and induction of apoptosis (caspase activation) when treated with a combination of TRAIL and BBR. This apoptosis is attenuated in cells overexpressing AMPKα-dominant negative (DN), suggesting an involvement of AMPK in mediating this. To understand the downstream targets and the mechanism involved, an apoptosis PCR array analysis was performed, which suggested induction of TNFRSF10B (DR5) gene expression by BBR. In addition, knockdown of DR5 expression attenuated TRAIL-BBR-induced apoptosis, suggesting DR5 to be a potential target of BBR in this apoptotic cascade. Future studies will include determining any crosstalk of AMPK in BBR signaling to modulate DR5 pathway. Our earlier studies also demonstrated an involvement of β-catenin in mediating cancer cell resistance. Stabilizing mutations and overexpression of β-catenin has been reported in many cancers, most profoundly in HCC leading to an activation of Wnt/β-catenin signaling. To understand any role of β-catenin in TRAIL-BBR-induced apoptosis, we determined the effect of BBR on β-catenin pathway. These revealed a significant attenuation of β-catenin protein expression by BBR in various HCC cells in a time and dose-dependent manner. BBR treatment also attenuated β-catenin/TCF-induced transcriptional activity, indicating antagonism of β-catenin pathway. Our studies indicate that combination of TRAIL and AMPK activator BBR might be an effective means of antagonizing β-catenin pathway and ameliorating TRAIL resistance via DR5 in advanced forms of cancer. Citation Format: Basabi Rana, Rong Ke, Kanchan Vishnoi, Navin Viswakarma, Subhasis Das, Ajay Rana. Elucidation of the signaling pathways that mediate berberine-induced effects in cancer cells [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 3322. doi:10.1158/1538-7445.AM2017-3322

Circulating tumor cells (CTCs) are known to disseminate from primary/metastatic tumors, survive in the circulation, and develop competency to colonize distant organs. Dissecting CTC heterogeneity is therefore crucial to provide improve prognostic and diagnostic tools (concept of “liquid biopsy”). However, the clinical significance of CTC heterogeneity is still unknown. We hypothesized that melanoma patient blood-derived, lineage-negative cells (CD45-/CD34-/CD73-/CD90-/CD105-) contain melanoma cell subpopulations (Melan A+, S100+, CD146+ and NG2+ CTCs) that evolve in vivo and achieve metastatic potency when reaching an organ-specific niche. We enriched Lin-negative cells from peripheral blood of melanoma patients clinically diagnosed with or without brain metastasis (MBM) by multi-parametric flow cytometry; and analyzed CTC heterogeneity in xenografts by longitudinal capturing and interrogation at the single-cell level (DEPArrayTM platform). First, we evaluated the presence of melanoma CTC subpopulations in Lin-negative cells and developed Lin-negative murine xenograft models. Human-cell derived melanoma metastasis in xenografts confirmed that CTCs require metastasis-potentiating factors present in the Lin-negative population. Second, we performed multi-parametric flow cytometry to isolate human-derived HLA+/Melan A+/- CTCs/DTCs population from murine blood and bone marrow at T0, Tmid and Tend phases of metastasis. We interrogated the differential levels of CTCs/DTCs in HLA+/Melan A+ cell population and established their neoplastic identity by genetic profiling and the expression of melanoma markers at single-cell level. Third, we dissected longitudinal transcriptional signatures of CTCs vs DTCs in association with MBM onset. We assessed the comprehensive EMT scores of CTCs and DTCs transcriptomics and identified the pathways associated with CTC/DTCs dynamic functional states depending upon melanoma progression. Collectively, this study provide important insights to identify novel EMT-related biomarkers of MBM progression. Further elucidation of these CTC biomarkers and properties may provide novel therapeutic strategies that can be clinically useful in melanoma patients which are yet to develop MBM. Citation Format: Monika Vishnoi, Haowen N. Liu, Debasish Boral, Wei Yin, Marc L. Sprouse, Jean P. Thiery, Isabella C. Glitza, Dario Marchetti. Translating CTCs for clinical use: melanoma patient-derived CTCs evolution in xenografts [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 1714. doi:10.1158/1538-7445.AM2017-1714

Patients with metastatic breast cancer have a five-year survival rate of 22% compared to 99% in patients with cancer localized to the breast. This disparity in clinical outcome is at least in part caused by the lack of diagnostic tools for early detection of metastatic recurrence. This can be especially valid for breast cancer patients having brain metastases (BCBM), 10-20% of whom are diagnosed only at autopsy. The early detection of circulating tumor cells (CTCs) in peripheral blood during the metastatic cascade provides a window of opportunity to prevent BCBM onset. However, to target CTCs causing BCBM, one must dissect mechanisms that allow CTCs to retain their brain metastatic potency while remaining dormant in circulation. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to maintain over time the potential for BCBM competence. Comprehensive analyses of breast cancer patient-derived CTC transcriptomes showed that CTC gene signatures closely relate to basal-like breast cancer cell lines rather than the molecular subtype of the primary tumor. However, unlike actively cycling cell lines, a large proportion of CTCs were mitotically dormant (Ki67-/PCNA-) with low overall mRNA expression and transcriptional activity. Upon systemic injection of patient-derived common lineage-depleted cells into immuno-compromised mice, the majority of CTCs were found at metastatic sites in a state of mitotic/metabolic dormancy. Second, dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by Serine139 phosphorylation status of gamma H2AX. Third, we discovered that the human telomere-associated protein RIF1, a mediator of alternative non-homologous end joining repair of DNA DSB interplaying with BRCA1/2, was significantly up-regulated in the Ki67-/PCNA- CTC population. RIF1/BRCA1 foci formation was confirmed in areas of DNA damage in these cells. Lastly, shRNA-mediated RIF1 knockdown promoted dormant cell populations to become more susceptible to UV and bleomycin-induced DNA damage while activating p38 and ERK stress-response pathways as well as their phosphorylation ratio. Moreover, RIF1 knockdown in MDA-MB231BR clone attenuated its metastatic competence to the brain. Collectively, these findings suggest that RIF1 regulates the genomic integrity of dormant CTCs allowing them to survive over time while in circulation. Further elucidation of RIF1-mediated CTC pathways and its dynamics with BRCA1/2 may provide novel strategies for therapeutic intervention. This can be clinically useful in breast cancer patients that are yet to develop overt BCBM. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Marc L. Sprouse, Jenny C. Chang, Jean Paul Thiery, Scamardo Antonio, David S. Hong, Dario Marchetti. Mechanisms underlying genomic integrity in breast cancer-derived dormant circulating tumor cells [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 1265. doi:10.1158/1538-7445.AM2017-1265

More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (>40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) as well as shRNA mediated RIF1 knockdown in HCC1954 and ZR-75-1 cell lines attenuated resistance of the dormant subset to UV and bleomycin treatment. Finally, RIF1 knockdown activated both p38 and pERK pathways albeit to varying degrees in multiple cell lines resulting in metastatic inefficiency in xenograft and syngeneic mouse models. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs and be a potential biomarker of breast cancer CTC survival while in circulation. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Marchetti D, Hong DS, Scamardo A. Maintenance of genomic integrity in dormant circulating tumor 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 P1-01-07.

Multiple studies concur that CTCs - the “seeds” of fatal metastasis - intravasate into the bloodstream throughout the early stages of cancer promoting generation of micro-metastatic reservoirs, some of which ultimately evolve to metastatic tumors. However, while it may take decades for CTCs to progress from cells in circulation to clinically detectable metastases, once detected, the tumor usually grows at an exponential rate. This difference in growth dynamics at two ends of the metastatic spectrum should also be reflected in the behavior of CTCs shed from these organ sites. Having a focus towards breast cancer brain-colonizing CTCs and their properties, we hypothesized that a gradual evolution of dormant CTCs into mitotically active CTCs with brain-metastatic potency is central to the progression of primary breast cancer to brain metastasis. We isolated CD44+/CD24- (stem-like) and Pan-Cytokeratin+ (epithelial) breast cancer CTCs from peripheral blood of two groups of breast cancer patients – five with brain metastases (BCBM) (MRI-detectable) and five without brain metastases (No BCBM). CTCs were isolated by multi-parametric flow cytometry and the expression of CTC-specific antigen was confirmed by immuno-cytochemistry and DEPArrayTM platform. Intra-cellular flow cytometry showed that No BCBM patients had 3-fold higher mitotically dormant (Ki67-) CTCs compared to BCBM patients. Whole genome expression arrays demonstrated low generalized transcriptional activity in CTCs suggestive of mitotic/metabolic dormancy. Comparison of CTC transcriptomes with ER+/PR+, HER2+ and triple negative breast cancers identified a unique CTC gene signature valid not only across the three molecular subtypes but also independent of the molecular subtype patients were initially diagnosed as. Pathway analyses predicted increased activation of pluripotency-related pathways along with decreased protein translational machinery and proliferative pathways in CTCs. Further, to dissect mechanisms of metastatic reactivation in the brain microenvironment, we compared the CTC transcriptome in BCBM vs No BCBM patients and identified a 126 gene signature potentiated in BCBM CTCs. Subsequent pathway analyses revealed upregulation of known CTC pathways like Notch along with novel hematopoietic and immune evasion networks. Cellular and functional annotations of cell migration and chemotaxis were significantly activated in BCBM along with pro-inflammatory (TNF, IL1β, NF-kB), immunomodulatory chemokines (CXCL8, CXCR4, CD86) and mitogenic growth factors (PDGF-BB). Lastly, lack of cell surface expression of Urokinase Plasminogen Activator Receptor (uPAR) and integrin beta-1 (int-β1) were used to identify CTCs in a state of metastatic dormancy. Whole genome sequencing showed significantly higher incidence of genomic mutations in proliferating CTCs compared to their dormant counterparts. We provide first time evidence of signaling pathways keenly implicated in breast cancer CTC biology based on comprehensive analyses of CTC transcriptomes. We discovered the existence of CTCs in a state of metabolic/mitotic dormancy and identified genomic instability as a switch for CTCs to revert to their proliferative phenotype. Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Hong DS, Scamardo AT, Marchetti D. The evolution of breast cancer circulating tumor cells mediating brain metastasis [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 P1-01-08.

Tumor relapse is a clinically relevant problem in breast cancer where patients are asymptomatic because disseminated cells appear to become dormant for periods longer than 20 years and are undetectable by current clinical tools. Uncovering phenotypes of circulating tumor cells (CTCs) - the “seeds” of intractable metastasis-offers the promise to dissect CTC heterogeneity in relation to metastatic competence, to predict biomarker assessment, and to significantly improve monitoring and treatment of cancer. However, little is known about CTC biology and how CTCs differ in their capacity to circulate while maintaining a metastatic potential. We hypothesized that EpCAM-negative breast cancer CTC subsets exist, and avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties. We collected peripheral blood of clinically diagnosed breast cancer patients with or without brain metastasis, and performed multiparametric flow cytometry to isolate EpCAM-negative CTC subsets with stem-cell properties (CD44+/CD24-), along with combinatorial expression of two neoplastic markers: urokinase plasminogen activator receptor (uPAR) and integrin beta1 (int β1). EpCAM-negative CTCs were further interrogated at a single-cell level employing DEPArray platform. Second, we were able to culture FACS-sorted CTC subsets, selected for six cell-surface expression markers (CD45-/EpCAM-negative/CD44+/CD24-/uPAR+/-/int β1+/-), as long-term in-vitro 3D CTC tumorspheres. Third, CTC subsets were interrogated for biomarker profiling and biological characteristics. We identified adhesive, proliferative and invasive properties of 3D CTC tumorspheres which were distinct per uPAR/int β1 combinatorial expression. Lastly, we performed next-generation whole-genome sequencing and mutation analyses to discover unique genomic signatures of uPAR/int β1 CTC subsets and verified as putative CTCs originally disseminated from primary breast tumor. Additional investigations are being pursued assessing the molecular and genomic characterization of uPAR/int β1 CTC subsets comprehensively. Clinical relevance of this research includes that this may enhance abilities to prospectively identify patients who may be at high-risk of developing breast cancer brain metastasis. Citation Format: Monika Vishnoi, Sirisha Peddibhotla, Wei Yin, Zhong Xue, Antonio T. Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Dissecting breast cancer dormant CTC phenotypes. [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 1530.

More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse. We isolated and characterized EpCAM-negative breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (>40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient in repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) attenuated resistance of the dormant subset to UV and bleomycin treatment. Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs. Further investigations are being pursued to assess RIF1 contributions to retain CTC LTI potential leading to CTC-driven metastasis. Citation Format: Debasish Boral, Haowen N. Liu, Wei Yin, Monika Vishnoi, Antonio Scamardo, Goldy C. George, David S. Hong, Dario Marchetti. Deciphering mechanisms of circulating tumor cells in breast cancer dormancy. [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 3143.

Uncovering phenotypes of patient-derived Circulating Tumor Cells (CTCs) offers the promise to dissect CTC heterogeneity in relation to metastatic competence, and to determine biomarkers of therapeutic utility for improved treatment. However, it is still unknown whether and how CTCs differ in their capacity to circulate while maintaining metastatic potential. Rates of CTC survival are highly variable, lasting less than few hours in some patients but in the order of decades in others. This can lead to many questions for yet unexplored mechanisms of CTCs responsible for dormancy, along with their properties and biomarker functionalities. We hypothesized that breast cancer CTC subsets possessing markers of pluripotency avoid organ arrest with extreme efficiency by the concomitant presence of quiescence and stem cell properties; and that expression of urokinase plasminogen activator receptor (uPAR) and beta-1 integrin (β1int), two biomarkers known to be directly implicated in tumor cell dormancy, are relevant in controlling the recurrence of breast cancer brain metastasis (BCBM). First, we isolated CTC subsets not expressing the epithelial cell adhesion molecule (EpCAM-negative CTCs), and characterized these subsets using DEPArrayTM, a new CTC platform able to dissect CTC heterogeneity at a single-cell level, thus interrogating the smallest functional unit of cancer. We captured EpCAM-negative/CD45-/CD44+/CD24- breast cancer CTC subsets that possessed combinatorial uPAR and β1int expression using multiparametric flow cytometry. Second, CTC subsets grew in vitro and were further characterized by DEPArrayTM. Markers expression was confirmed by confocal microscopy with subsets possessing a specific breast cancer gene profiling. Third, EpCAM-negative CTC subsets (uPAR+/β1int+ and uPAR-/β1int-) were interrogated for human embryonic stem cell markers by RT2 PCR arrays. Gene expression profiling was consistently distinct among uPAR+/β1int+ vs. uPAR-/β1int- CTC subsets and dependent upon patients’ BCBM status: expression of genes implicated in cell cycle progression (e.g., CDK42, CDK1), angiogenesis (e.g., FGF-2), and pluripotency (e.g., KLF4) was >30-fold higher than controls. Third, CTC subsets gene patterns isolated from patients with BCBM possessed RT2 profiles that were strikingly distinct from ones derived from patients with no BCBM. Of note, gene expression for RIF-1, a protein that counteracts actions of the breast cancer suppressor BRCA1, was highest (>50-fold) with distinct RIF-1 nuclear patterns in BCBM CTC subsets. In summary, we have linked EpCAM-negative uPAR/β1int CTC subsets and their properties to clinical BCBM; and will assess the therapeutic inhibition of uPAR/β1int CTC biomarkers on BCBM development and its timing. Deciphering the relevance of uPAR/β1int as key CTC biomarkers of dormancy vs. metastatic competence will elucidate CTC mechanisms responsible for BCBM onset. Citation Format: Sirisha Peddibhotla, Monika Vishnoi, Wei Yin, Yizhen Chen, Antonio Scamardo, David Hong, Dario Marchetti. Dissecting CTC phenotypes: insights into mechanisms of breast cancer dormancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 363. doi:10.1158/1538-7445.AM2015-363

Background & Objectives: Persistent infection of high-risk human papillomaviruses (HR-HPV) is a key etiological factor responsible for the development of cervical cancer. Deregulated expression of two viral oncoproteins E6 and E7 drive oncogenic transformation and tumour progression which may occur upon targeted infection of specific basal cervical cells with stem cell properties. However, how oncoproteins impact on putative CSCs, the stem cell signaling and their downstream functions, is not known. In this study, we have identified and characterized cervical cancer stem-like cells and unraveled the crosstalk between HPV oncoproteins and stem cell signaling that allowed maintenance of stemness in cancer cells. Experimental Procedure: Cervical cancer stem-like cells (CaCxSLCs) were isolated and enriched by sequential gating from HPV+ve and HPV-ve human cervical cancer cell lines (SiHa, HeLa and C33a) using a set of functional and phenotypic markers (ABCG2, CD49f, CD71, CD133) in defined conditioned media (DCM) with intermittent culturing. CaCxSLCs were also assessed for their cervicosphere forming ability, exclusion of DCV dye for generating SP cells, self renewability and quiescenceness. Moreover, differential expression level and DNA-binding activity of CSL-specific Notch1 and its downstream targets in CaCxSLCs cells along with HPVE6 specific siRNA mediated gene silencing was evaluated by gel retardation assay, immunobloting qRT-PCR followed by in vivo tumor-initiating capacity in athymic nude mice. Results: CaCxSLCs cells isolated from HPV+ve cells showed high expression of CD49f, Lrig1, Nanog, Sox2, Oct4, ABCG2 and low Notch1 DNA binding activity as compared to non- CaCxSLCs cells. Enriched CaCxSLCs were also found to be more tumorigenic and showed high expression of HPVE6, HES1, CD133 transcripts with high percentage of SP cells. CaCxSLCs differed from their parental/non-CaCxSLCs as they overexpressed E6 oncogene. Knocking down of HPVE6 by RNA interference resulted in abolition of spheroid formation through downregulation of Hes1 and induction of re-differentiation while interference of Hes-1 expression resulted in reduced cervicosphere formation through down regulation of E6 and Nanog suggesting loss of self-renewing ability. Conclusions: Our findings suggest a potential regulatory role of HPVE6 in induction and maintenance of cervical cancer stem-like cells through Hes1 expression which could be utilized as a putative target for designing drugs that can promote differentiation of cancer stem cells making the therapy most effective. Note: This abstract was not presented at the meeting. Citation Format: Bhudev C. Das, Abhishek Tyagi, Kanchan Vishnoi, Sutapa Mahata, Gaurav Verma, Alok C. Bharti. Role of HPVE6 oncoprotein in the maintenance of cervical cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2015-4079