Ila Datar

SWI/SNF Enzymes Promote SOX10- Mediated Activation of Myelin Gene Expression

SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to directly activate genes that encode components of peripheral myelin.

RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration

Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of-function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.

RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis.

A Micro-RNA Connection in BRaf(V600E)-Mediated Premature Senescence of Human Melanocytes.

Recent high-throughput-sequencing of the cancer genome has identified oncogenic mutations in BRaf genetic locus as one of the critical events in melanomagenesis. In normal cells, the activity of BRaf is tightly regulated. Gain-of-function mutations like those identified in melanoma frequently lead to enhanced cell-survival and unrestrained growth. The activating mutation of BRaf will also induce the cells to senesce. However, the mechanism by which the oncogenic BRaf induces the senescent barrier remains poorly defined. microRNAs have regulatory functions toward the expression of genes that are important in carcinogenesis. Here we show that expression of several microRNAs is altered when the oncogenic version of BRaf is introduced in cultured primary melanocytes and these cells undergo premature cellular senescence. These include eight microRNAs whose expression rates are significantly stimulated and three that are repressed. While most of the induced microRNAs have documented negative effects on cell cycle progression, one of the repressed microRNAs has proven oncogenic functions. Ectopic expression of some of these induced microRNAs increased the expression of senescence markers and induced growth arrest and senescence in primary melanocytes. Taken together, our results suggest that the change in microRNA expression rates may play a vital role in senescence induced by the oncogenic BRaf.

Abstract 5657: Objective measurement and significance of VISTA (PD-1H) expression in non-small cell lung cancer (NSCLC)

Introduction: VISTA (PD-1H) is a member of the B7 family of immune co-regulatory molecules and has been proposed as a novel anti-cancer immunotherapy target. The intra- and extracellular domains of VISTA show homology to PD-1 and PD-L1, respectively, suggesting a role in anti-tumor immune evasion. The expression of VISTA, its association with PD-1 axis components and biological role in human NSCLC are unknown. Methods: Using multiplex quantitative immunofluorescence (QIF), we simultaneously measured the levels of VISTA (clone D1L2G, CST), PD-L1 (clone 405.9A11, CST) and PD-1 (clone EH33, CST) protein in 732 stage I-IV NSCLCs from 3 retrospective collections represented in tissue microarray format (cohort #1 [n=297, Yale], cohort #2 [n=329, Greece]; and cohort #3 [n=106, Yale]). To evaluate the tumor tissue distribution, VISTA was also selectively measured in cytokeratin+ tumor cells, CD3+ T-cells, CD4+ T-helper cells, CD8+ cytotoxic T-cells and CD20+ B-lymphocytes. Associations between the marker levels, clinico-pathological-molecular variables and survival were studied. Results: VISTA protein was detected in all NSCLCs, showed a membranous staining pattern and was localized predominantly in the tumor cells in 27.4% of cases; and in the stromal compartment in 98.5%. Although VISTA was detected in all major tumor infiltrating lymphocyte (TIL) subsets, the signal was higher in CD20+ B-cells than in CD3+ T-lymphocytes (P Conclusion: VISTA is expressed in the majority of NSCLCs and shows differential distribution in tumor/stromal cells and TIL subsets, suggesting a complex function as a ligand and receptor. Elevated expression of VISTA in NSCLC is associated with increased PD-1 axis targets and cytotoxic T-cell density, indicating its possible modulation by pro-inflammatory signals. Our results support VISTA as a candidate target for anti-cancer immunotherapy in NSCLC alone or in combination with PD-1 axis blockers. Citation Format: Franz Villarroel-Espindola, Ila J. Datar, Vamsidhar Velcheti, David L. Rimm, Roy S. Herbst, Kurt A. Schalper. Objective measurement and significance of VISTA (PD-1H) expression in non-small cell lung cancer (NSCLC) [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 5657. doi:10.1158/1538-7445.AM2017-5657

Abstract 5600: Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC)

Introduction: The ineffective anti-tumor immune response is characterized by increased immune suppressive signals in the tumor microenvironment. In particular, T-cells recognizing tumor antigens can express diverse immune inhibitory receptors mediating lymphocyte inactivation and limiting tumor rejection. Blockade of these receptors such as PD-1 induces prominent clinical benefit in patients with NSCLC. However, the expression and significance of additional potentially actionable immune inhibitory receptors in lung cancer is poorly understood. Methods: After careful validation of assays and using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (clone EH33, CST), LAG-3 (Clone 17B4, Abcam) and TIM-3 (clone D5D5R, CST) in 698 stages I-IV formalin-fixed paraffin embedded (FFPE) lung carcinomas represented in three tissue microarrays (cohort #1 [Yale n=186], cohort #2 [Yale n=192, and cohort #3 [Greece n=320]). We also included a collection of lung adenocarcinomas with molecular annotation (cohort #4 [Yale n=106]). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers and with major clinico-pathological variables, driver mutations and survival were studied. Results: All the targets were detected predominantly in CD3+ T-cells with membranous staining. Expression of PD-1, LAG-3 and TIM-3 in T-cells across all NSCLC cohorts was 68.7%, 39.7% and 55.8%, respectively. Elevated levels of PD-1, LAG-3 or TIM-3 were significantly associated with increased tumor infiltrating lymphocytes and with the co-expression of one or more of the other inhibitory receptors (P Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in NSCLC, show frequent co-expression and association with elevated CD3+ T-cells. Our results support the biological role of PD-1, LAG-3 and TIM-3 in NSCLC and suggest co-activation of these immune inhibitory pathways in a proportion of cases. Modulation of these receptors could enhance the anti-tumor immune response in lung cancer. Citation Format: Ila J. Datar, Jun Wang, Nikita Mani, Franz Villarroel-Espindola, Patrick Ryan, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, David L. Rimm, Leiping Chen, Roy Herbst, Kurt Schalper. Simultaneous measurement and clinical significance of PD-1, LAG-3 and TIM-3 in non-small cell lung cancer (NSCLC) [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 5600. doi:10.1158/1538-7445.AM2017-5600

Abstract 1635: Multiplexed analysis of fixed tumor tissues using imaging mass cytometry

Introduction: Comprehensive evaluation of the tumor microenvironment with preservation of spatial context requires simultaneous in situ detection of multiple targets. Current fluorescence-based methods can accommodate up to 5-6 distinct markers and can be applied to formalin fixed, paraffin embedded tissue (FFPE). Imaging mass cytometry uses metal-conjugated antibodies and mass spectrometry to perform highly multiparametric, quantitative measurement of protein targets within tissue sections providing unprecedented detail of quantitative tissue labeling. To date, the IMC technology has not been widely available and its performance has not yet been compared with other validated methods. Methods: Using multiplexed quantitative immunofluorescence (QIF, AQUA/Genoptix) and imaging mass cytometry (IMC, Fluidigm) we validated and optimized metal-conjugated assays to detect HER2 (29D8-CST, 176Yb), CD3 (DAKO, 170Er), Ki-67 (B56-BD biosciences, 168Er), Histone H3 (D1H2-CST, 176Yb), pancytokeratin (C11-Biorad, 162Dy), Vimentin (RV202-BD Pharmingen, 156Gd), LipoR (Fluidigm, 115In) and DNA/nuclei (DNA intercalator, Fluidigm, 191/193Ir). The markers were applied to FFPE samples from tumor cell lines, human tonsil, lymph node; and breast and lung carcinomas represented in tissue microarray format. Data was visualized using MCD viewer and ImageJ software and results from QIF and IMC were compared. Results: Metal conjugation did not affect the performance of the primary antibodies studied here. The staining patterns of the epithelial/tumor and stromal compartments evaluated using the IMC platform and QIF were comparable. HER2 signal was higher in tumors and cell lines harboring HER2 amplification by FISH/IHC. The CD3 signal was higher in T-cell lymphoma cells and in the inter-follicular areas of human tonsil and lymph nodes. Ki-67 signal was nuclear, co-localized with Histone H3 and showed positivity in lymphoid germinal centers, cytokeratin-positive carcinoma cells and CD3+ tumor infiltrating lymphocytes. Results obtained using the IMC at different time points in serial tissue sections were comparable (P Conclusion: Quantitative and reproducible measurement of immune and non-immune targets with spatial resolution using the IMC platform is feasible in FFPE breast and lung carcinomas and the results are comparable to multiplexed QIF. Expansion of the current panel to include >30 markers for immune cell phenotypes/function and tumor markers is ongoing. Citation Format: Franz Villarroel-Espindola, Daniel Carvajal-Hausdorf, Ila Datar, Amanda Esch, Narges Rashidi, Ala Nassar, Shelly Ren, Ruth R. Montgomery, Roy S. Herbst, David L. Rimm, Kurt A. Schalper. Multiplexed analysis of fixed tumor tissues using imaging mass cytometry [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 1635. doi:10.1158/1538-7445.AM2017-1635