Sanket H. Shah

Identification of cancer associated fibroblasts in circulating blood from patients with metastatic breast cancer

Metastasis is facilitated by cancer-associated fibroblasts (CAF) in the tumor microenvironment through mechanisms yet to be elucidated. In this study, we used a size-based microfilter technology developed by our group to examine whether circulating CAF identified by FAP and α-SMA co-expression (cCAF) could be distinguished in the peripheral blood of patients with metastatic breast cancer. In a pilot study of patients with breast cancer, we detected the presence of cCAFs in 30/34 (88%) patients with metastatic disease (MET group) and in 3/13 (23%) patients with localized breast cancer (LOC group) with long-term disease-free survival. No cCAFs as defined were detected in healthy donors. Further, both cCAF and circulating tumor cells (CTC) were significantly greater in the MET group compared with the LOC group. Thus, the presence of cCAF was associated with clinical metastasis, suggesting that cCAF may complement CTC as a clinically relevant biomarker in metastatic breast cancer.

Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as “dissociated tumor (DT) cells.” In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.

Hierarchical paracrine interaction of breast cancer associated fibroblasts with cancer cells via hMAPK-microRNAs to drive ER-negative breast cancer phenotype

Multiple juxtacrine and paracrine interactions occur between cancer cells and non-cancer cells of the tumor microenvironment (TME) that direct tumor progression. Cancer Associated Fibroblasts (CAFs) are an integral component of the TME, and the majority of breast tumor stroma is comprised of CAFs. Heterotypic interactions between cancer cells and non-cancer cells of the TME occur via soluble agents, including cytokines, hormones, growth factors, and secreted microRNAs. We previously identified a microRNA signature indicative of hyperactive MAPK signaling (hMAPK-miRNA signature) that significantly associated with reduced recurrence-free and overall survival. Here we report that the hMAPK-miRNA signature associates with a high metric of stromal cell infiltrate, and we investigate the role of microRNAs, particularly hMAPK-microRNAs, secreted by CAFs on estrogen receptor (ER) expression in breast cancer cells. ER-positive MCF-7/ltE2- cells were treated with conditioned media (CM) from CAFs derived from breast cancers of different PAM50 subtypes (CAFBAS, CAFHER2, and CAFLA). CAF CM isolated specifically from ER-negative primary breast tumors led to ER repression in vitro. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of CAF-secreted exosomes in CM and the uptake of these exosomes by the ER+ MCF-7/ltE2- cells. Differentially expressed microRNAs in CAF CM as well as in MCF-7/ltE2- cells treated with this CM were identified. Knockdown of miR-221/222 in CAFBAS resulted in knockdown of miR221/222 levels in the conditioned media and the CM from CAFBAS; miR221/222 knockdown rescued ER repression in ER-positive cell lines treated with CAFBAS-CM. Collectively, our results demonstrate that CAF-secreted microRNAs are directly involved in ER-repression, and may contribute to the MAPK-induced ER repression in breast cancer cells.

Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration Type 1 diabetes

Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.

Inhibition of novel GCN5-ATM axis restricts the onset of acquired drug resistance in leukemia: GCN5-ATM axis in leukemia resistance

Leukemia is majorly treated by topoisomerase inhibitors that induce DNA double strand breaks (DSB) resulting in cell death. Consequently, modulation of DSB repair pathway renders leukemic cells resistant to therapy. As we do not fully understand the regulation of DSB repair acquired by resistant cells, targeting these cells has been a challenge. Here we investigated the regulation of DSB repair pathway in early drug resistant population (EDRP) and late drug resistant population (LDRP). We found that doxorubicin induced equal DSBs in parent and EDRP cells; however, cell death is induced only in the parent cells. Further analysis revealed that EDRP cells acquire relaxed chromatin via upregulation of lysine acetyl transferase KAT2A (GCN5). Drug treatment induces GCN5 interaction with ATM facilitating its recruitment to DSB sites. Hyperactivated ATM maximize H2AX, NBS1, BRCA1, Chk2, and Mcl‐1 activation, accelerating DNA repair and survival of EDRP cells. Consequently, inhibition of GCN5 significantly reduces ATM activation and survival of EDRP cells. Contrary to EDRP, doxorubicin failed to induce DSBs in LDRP because of reduced drug uptake and downregulation of TOP2β. Accordingly, ATM inhibition prior to doxorubicin treatment completely eliminated EDRP but not LDRP. Furthermore, baseline AML samples (n = 44) showed significantly higher GCN5 at mRNA and protein levels in MRD positive compared to MRD negative samples. Additionally, meta‐analysis (n = 221) showed high GCN5 expression correlates with poor overall survival. Together, these results provide important insights into the molecular mechanism specific to EDRP and will have implications for the development of novel therapeutics for AML.

Abstract 3174: GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia

Abstract Introduction- Resistance to therapeutics targeting topoisomerase 2 is a major problem in the treatment of leukemia. Cells which survive and give rise to relapsed leukemia are known to modulate different pathways like downregulation of drug target, reduced drug accumulation, and improved DNA repair; ultimately leading to survival of drug resistant cells. Here, we wanted to identify the earliest detectable changes occurring when cells become resistant to topoisomerase 2 inhibitors. Methodology- We generated early and late stage drug (doxorubicin) resistant leukemic sub cell line from K562 and THP-1 parent cells, HL-60/MX2 resistant sub cell line of HL60 was also used for this study. Molecular changes were analyzed by electron microscopy, quantitative PCR, western blotting, immunofluorescence, flow cytometry, molecular inhibitors and MTT assay. Results were further confirmed in blast cells of AML (n=44) patient samples collected for this study. Furthermore, meta-analysis for survival was done from microarray expression data of 221 patient samples using PrognoScan. Results- We mapped molecular changes acquired by leukemic cells during evolution from Early Drug Resistant Population (EDRP) to Late Drug Resistant Population (LDRP). We found unlike LDRP, EDRP cells do not possess known bona fide drug-resistance mechanisms namely limited drug accumulation, reduced DNA damage or expression of drug target. Instead they survive by acquiring ‘poised epigenetic state’ that enhanced their DNA repair. Mechanistically, GCN5, a histone acetyl transferase get selectively upregulated in EDRP cells mediating higher H4K16 acetylation levels and consequent euchromatin state of EDRP. Upon Doxorubicin treatment, H4K16ac facilitate higher ATM recruitment and activation causing efficient activation of H2AX, NBS1, BRCA1, Chk2 and Mcl-1, accelerating DNA repair and survival of EDRP cells. Inhibition of GCN5 with Doxorubicin treatment significantly reduces H4K16ac levels, ATM recruitment and cell survival of EDRP cells. Similarly, ATM inhibition along with Doxorubicin completely ablates EDRP but not LDRP. Furthermore, baseline AML patient samples (n=44) showed significantly higher GCN5 expression in MRD positive compared to MRD negative samples. Additionally, meta-analysis of 221 AML patients showed increased GCN5 expression associates with poor survival of AML patients. Conclusion- We identify GCN5 expression as marker that defines onset of resistance in leukemia and GCN5 mediated ATM activation via H4K16ac as a novel non-genetic route facilitating EDRP cell survival with enhanced DNA repair. These data also highlight the clinical relevance of targeting GCN5 and ATM during early resistance to prevent emergence of difficult to treat stable diverse resistance in leukemia. Citation Format: Sameer J. Salunkhe, Jyothi Nair, Ekjot Kaur, Neha Chaoudhary, Sanket Shah, Ketaki Patkar, Dev Anand, Navin Khattry, Syed K. Hasan, Shilpee Dutt. GCN5 regulates ATM mediated DNA repair responsible for onset of acquired resistance in leukemia [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 3174. doi:10.1158/1538-7445.AM2017-3174

Abstract 4396: Circulating CAFs and CAF-secreted factors may be indicative of breast cancer metastasis

Tumor metastasis is the main cause of breast cancer mortality. Increasing evidence demonstrates stromal cells play pivotal roles in promoting breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that promote breast cancer progression and metastasis; one of these factors is SDF-1 (CXCL12), a soluble chemokine that is promotes chemotaxis and motility, and facilitates cancer cell motility and angiogenesis. CAFs also secrete soluble factors that activate ERK 1/2 MAPK signaling in breast cancer cells, which has been shown to promote loss of estrogen receptor (ER) in luminal breast cancer cells. Hyperactivation of MAPK signaling (hMAPK) also associates with aggressive, basal-like and HER2-positive breast cancer and poor prognosis. Recently, we identified a patient-derived hMAPK-microRNA signature indicative of poor clinical outcome that contains microRNAs known to regulate breast cancer associated genes. The vast majority of ER- breast cancers display this microRNA signature, as do a subset of ER+ breast cancers with poorer clinical outcome. We also discovered that the breast cancers that exhibit this microRNA signature display high stromal and immune infiltrate scores, suggesting that breast cancer stroma provides important contributions to this microRNA signature and the poor clinical outcomes associated with it. To study the role of CAFs and CAF-secreted factors in breast cancer progression and metastasis, we established primary breast CAF lines from ‘indolent’ breast cancers (Luminal A), and from ‘aggressive’ breast cancers (ER-/HER2 amplified; triple negative). We have demonstrated that these CAFs differentially express several members of the hMAPK-microRNA signature compared to cultured primary breast cancer cells, supporting the contribution of stroma to the signature. Importantly hMAPK-microRNAs secreted from “aggressive” CAFs can be taken up by breast cancer cells, whereupon they repress their targets. Normal human mammary fibroblasts (HMFs) and ‘indolent’ CAFs do not secrete these microRNAs. We identified a novel class of circulating cells in the blood of breast cancer patients with metastases -CAFs (cCAFs). Patients without metastases did not have these cCAFs - suggesting cCAFs may be “aggressive” CAFs that facilitate breast cancer metastasis. Patients with overt metastasis and elevated counts of cCAFs have significantly higher levels of circulating SDF-1 in their plasma, as well as differential circulating microRNAs, and specifically hMAPK-microRNAs also found secreted by “aggressive” CAFs. Our results suggest there is a hierarchy of CAFs whereby “aggressive” tumors establish “aggressive” CAFs to facilitate metastasis. We also establish a clear link between circulating CAFs and CAF-secreted factors such as SDF-1 and microRNAs with breast cancer metastasis. Citation Format: Sanket H. Shah, Phil Miller, Leah Machlin, Kelsie Medina-Saenz, Ritesh Parajuli, Marc E. Lippman, Dorraya El-Ashry. Circulating CAFs and CAF-secreted factors may be indicative of breast cancer metastasis. [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 4396.

Abstract B39: Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer

Over the past decade an accumulating body of evidence has demonstrated the pivotal role of stromal cells in promoting breast cancer progression. In breast cancer, the stroma is mainly comprised of carcinoma-associated fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that are known to activate MAPK pathway. We have previously established that hyperactivation of MAPK in breast cancer cells leads to repression of estrogen receptor (ER) expression. Aberrant mitogenic signaling also affects expression of microRNAs (miRNAs). We have recently identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK-miRNAs). Most ER- breast cancers and a population of ER+ breast cancers are identified by this signature, and these hMAPK-miRNA tumors exhibit significantly decreased disease-free survival and overall survival compared to tumors that are not hMAPK-miRNA. Some of these hMAPK-miRNAs have been established as targeting ER. To further investigate the contribution of CAFs in activation of MAPK signaling in cancer cells, we isolated CAF cell cultures from three different primary breast tumors: CAF19 (from a luminal A tumor), CAF21 (from a Her-2 like tumor) and CAF23 (from a basal like tumor) and tumor cell cultures (DTs) from 5 different primary breast tumors. There is differential expression of hMAPK miRNAs between these CAFs and DTs, including those that target ER. Thus, we propose that interactions between cancer and stromal cells lead to activation of MAPK signaling in the cancer cell and aberrant miRNA expression within the TME that contribute to the biology of ER-negative tumors. To further investigate the connection between hMAPK-miRNAs and stroma, we analysed TCGA breast cancer datasets and found that hMAPK-miRNAs are significantly associated with tumors that possess high stromal scores. Co-culture of CAFs with ER+ breast cancer cells, as well as treatment of ER+ breast cancer cells with conditioned media (CM) from CAFs, induces both MAPK activation and altered expression of miRNAs, specifically MAPK miRNAs within the breast cancer cells. Treatment of ER+ breast cancer cells with CM from CAFs represses both ER protein and mRNA expression, and importantly, this ER repression is specific to CAFs isolated from ER-negative breast tumors (CAF21 and CAF23) compared to the CAFs obtained from ER-positive tumors (CAF19). Additionally, CM from CAF21 and CAF23 caused repression of an ER 39UTR reporter construct transfected into ER+ breast cancer cells, indicating a role for miRNAs either secreted into the CM or induced in the cancer cell in repressing ER. To investigate a role for hMAPK-miRNAs secreted by CAFs in mediating ER repression, nanostring analysis of miRNAs isolated from ER+ breast cancer cells treated with CAF CM was performed and differentially expressed miRNAs between CM from CAFs obtained from ER-positive (DT19) vs ER-negative tumors (DT21 and DT23) were identified. qPCR analysis of conditioned media also confirmed the presence of hMAPK-miRNAs in CAF CM. Collectively, these data indicate a role for CAFs in regulating hMAPK-miRNAs, and thus in establishing an ER-negative breast cancer phenotype, and importantly, differences between CAFs from ER-negative as compared to ER-positive tumors in this role. They further suggest that hMAPK-miRNAs secreted from CAFs participate in the induction of the ER-negative phenotype induced by MAPK. Citation Format: Sanket H. Shah, Phil Miller, Emilio Issa, Katherine Drews-Elger, Dorraya El-Ashry. Stromal miRNAs as mediators of mitogen activated protein kinase-induced estrogen repression in breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B39. doi:10.1158/1538-7445.CHTME14-B39

Abstract 3317: Radiation induced multinucleated giant cells: A novel therapeutic target to prevent survival and relapse of glioblastoma

In this study, we address the fundamental issue of radiation resistance in Glioblastoma Multiforme (GBM) a highly malignant form of brain tumor. However, because of the unavailability of multiple biopsies of GBM patients after treatment, we are limited in our understanding of resistance mechanisms. To circumvent this problem we recapitulated clinical scenario of GBM resistance in a cellular model developed from fresh naive primary GBM patient samples and cell lines. Using our model we show that upon lethal dose of radiation, small percentage of GBM cells survive, are non-apoptotic and transiently become non- proliferative. They are arrested in G2/M phase of cell cycle mediated by inhibitory phosphorylation of Cdk1(Y15) and p21. Surprisingly, we find although non -proliferative, these cells are highly motile and undergo homotypic cell-cell fusions at high frequency to form multinucleated and giant cells (MNGCs). Cell fusion leads to the induction of senescence as seen by β-galactosidase staining and accordingly high expression of SASPs (senescence associated secretory proteins like GM-CSF, SCF, IL-6 and IL-8) is also observed. SASPs provide survival and proliferation signals to the cells in an autocrine manner. Accordingly, we find enhanced expression of anti-apoptotic genes BIRC3 and Bcl-xL and unaltered levels of Bax (pro-apoptotic protein) mRNA in the resistant cells. MNGCs also activate AKT, which is a survival signal and a central convergence node of signals downstream to SASPs. AKT activation along with Bcl-xL is reported to regulate apoptosis synergistically. Additionally, resistant cells also undergo active DNA damage repair mediated by sensory kinases ATM and ATR. Interestingly, irrespective of the phase of cell cycle, they adopt Non Homologous End Joining (NHEJ) as their pathway of choice to repair their double strand breaks in DNA. Since we find increased presence of H3K36me2 marks known to modulate NHEJ, a possible link between NHEJ and this modification in GBM is worth exploring. Finally, unlike previous reports we demonstrate that MNGCs do not die of mitotic catastrophe instead escape senescence, undergo normal cell division and proliferation giving rise to mono-nucleated relapse cells with unaltered ploidy compared to parent cells. Importantly, we also show the potential ability to prevent the relapse in Glioma by disrupting the non-proliferative state of radiation resistant cells using mitotic inducer (MK1775) and selectively ablating growth of MNGCs by cytokinesis inhibitor. In summary, our data provides a mechanistic explanation for clinical observations and novel insights into an unexplored multi-step process of radiation survival and relapse in Glioblastoma We identified homotypic cell fusions of resistant Glioma cells as a novel non-genetic mechanism to sustain survival and relapse and generate rationale for novel combinatorial targeted therapies. Citation Format: Ekjot Kaur, Jacinth Rajendra, Sanket Shah, Jyothi Nair, Ankit Khushwaha, Aliasgar Moiyadi, Shilpee Dutt. Radiation induced multinucleated giant cells: A novel therapeutic target to prevent survival and relapse of glioblastoma. [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 3317. doi:10.1158/1538-7445.AM2015-3317

Abstract B108: Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs

The tumor microenvironment (TME) has been shown to play a vital role in tumor development and progression. Carcinoma associated fibroblasts (CAFs) form a very essential component of the TME. In fact, the TME in different types of carcinomas, particularly carcinomas of the breast, is comprised mainly of fibroblasts. CAFs in breast cancer TME secrete cytokines and growth factors that are known to activate a multitude of signaling pathways in breast cancer cells. One of these pathways is the MAPK pathway, a signaling pathway activated downstream of ERBB receptor tyrosine kinase family members. We have previously demonstrated that activation of the MAPK pathway represses estrogen receptor (ER) expression leading to an ER-negative phenotype in breast cancer. We have generated dissociated CAF cell cultures from ER+, ER-/Her2+, and triple negative (TN) primary breast tumors. We have characterized these CAFs on the basis of fibroblast markers, epithelial markers, microarray analysis, soft agar assay and in vivo tumorigenecity studies. Our results show that these CAF populations from primary breast tumors are pure populations of CAF cell cultures. These CAFs provide a model that allows us to examine the role of CAF interaction with ER+ breast cancer cells regarding activation of MAPK and subsequent repression of ER expression. We demonstrate that treatment of the ER+ MCF-7 cell line with conditioned media (CM) from the CAFs results in transient activation of MAPK signaling and subsequent repression of ER. Continuous exposure of ER+ breast cancer cells to soluble factors from CAFs also results in activation of MAPK and down-regulation of ER expression. Importantly, CM from human mammary fibroblasts (HMFs) and CAFs generated from an ER+ tumor do not down-regulate ER expression. Gene expression analysis and cytokine arrays indicate key differences in gene and cytokine expression between HMFs and the ER+ tumor CAFs compared to the two CAFs generated from ER- tumors. We have previously identified a miRNA signature associated with hyperactivation of MAPK signaling (hMAPK). This signature identifies the majority of ER- breast cancers as well as a population of ER+ breast cancers that express lower levels of ER. Like the ER- breast cancers identified by this signature, ER+ cancers identified by this signature exhibit gene expression patterns indicative of activated MAPK signaling, and exhibit significantly increased disease recurrence and significantly reduced disease survival. Here we show that breast tumors cancers bearing this hMAPK-miRNA signature have elevated expression of numerous stromal markers associated with poor clinical outcome, as well as a number of microRNAs that are differentially expressed between normal human mammary fibroblasts (HMFs) and mammary tumor carcinoma CAFs. Some of these miRNAs have been established as targeting ER. Using reporter constructs to investigate microRNA regulation of ER, we have observed that treatment of ER+ breast cancer cells with CM from ER- CAFs results in enhanced microRNA-dependent repression of ER. These data are suggestive of a role for CAF secreted factors in activating MAPK in breast cancer cells leading to repression of ER. In addition, CAFs are able to increase the expression of hMAPK-miRNAs that can down-regulate ER expression either via the activation of MAPK in the cancer cell or secreted directly from the CAFs. We hypothesize that CAFs may contribute to an ER-negative phenotype in breast cancer cells not only by activating MAPK signaling in the tumor cells via secretion of growth factors, but also potentially by transfer of MAPK-regulated miRNAs to the breast cancer cells as well. Citation Format: Sanket H. Shah, Philip Miller, Katherine Drews-Elger, Joeli Brinkman, Stefania Lairet, Alana Steinberg, Dorraya El-Ashry. Cancer-associated fibroblasts contribute to establishment of ER-negative breast cancer phenotype through secreted factors and miRNAs. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B108.