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BRG1 is one of two catalytic subunits of the SWI/SNF ATP-dependent chromatin-remodeling complex. In cancer, it has been hypothesized that BRG1 acts as a tumor suppressor. Further study has shown that, under certain circumstances, BRG1 acts as an oncogene. Targeted knockout of BRG1 has proven successful in most cancers in suppressing tumor growth and proliferation. Furthermore, BRG1 effects cancer proliferation in oncogenic KRAS mutated cancers, with varying directionality. Thus, dissecting BRG1’s interaction with various cellular pathways can highlight possible intermediates that can facilitate the design of different treatment methods, including BRG1 inhibition. Autophagy and apoptosis are two important cellular responses to stress. BRG1 plays a direct role in autophagy and apoptosis and likely promotes autophagy and suppresses apoptosis, supporting unfettered cancer growth. PRMT5 inhibits transcription by interacting with ATP-dependent chromatin remodeling complexes, such as SWI/SNF. When PRMT5 associates with the SWI/SNF complex, including BRG1, it represses tumor suppressor genes. The Ras/Raf/MAPK/ERK1/2 pathway in cancers is a signal transduction pathway involved in the transcription of genes related to cancer survival. BRG1 has been shown to effect KRAS-driven cancer growth. BRG1 associates with several proteins within the signal transduction pathway. In this review, we analyze BRG1 as a promising target for cancer inhibition and possible synergy with other cancer treatments.

Background: Breast cancer cells use tumor microenvironment of metastasis (TMEM) doorways as portals for hematogenous dissemination to distant sites. Each TMEM doorway is composed of one perivascular macrophage in direct contact with one endothelial cell and one Mena expressing cancer cell. TMEM density is prognostic of metastatic outcome in breast cancer patients. Since the development of metastases requires tumor initiating properties in cancer cells, and we recently demonstrated that cancer cells expressing cancer stem cell biosensors and other stem cell markers accumulate around TMEM (Sharma et al 2020 AACR abstract), we wanted to assess the relationship between stem cell marker expression and TMEM in mouse and human breast cancer. Methods: We evaluated the distribution of cancer stem cells relative to TMEM doorways in MDA-MB-231breast cancer cell xenografts that express a reporter (SORE6) and in transgenic PyMT mammary carcinomas using Sox9 as marker of stemness. We also evaluated the correlation of the percentage of cancer cells expressing stem cell markers (CD44high/CD24low, CD133, ALDH, and Sox9) with TMEM and the distribution of cancer cells expressing stem cell markers relative to TMEM in 49 breast cancers collected from patients. The stem cell markers were assessed by flow cytometry, fluorescence in situ hybridization (FISH), and qRT-PCR in Fine Needle Aspirates (FNA), as well as in fixed tissues by immunofluorescence assay. TMEM density was assessed in corresponding formalin-fixed and paraffin-embedded (FFPE) tumors by immunohistochemistry. The correlation of TMEM with stemness and the distance analysis of stem cells relative to TMEM was evaluated by Spearman's and Pearson's rank correlation respectively. Results: We observed a 7- and 3.4-fold enrichment of cancer stem cells (CSCs) close to TMEM ( Conclusion: The density of TMEM doorways positively correlates with the number of cancer stem cells in human breast cancer. Moreover, stem cells accumulate around TMEM consistent with the findings that TMEM doorways represent an educational niche for stemness in breast cancer. Citation Format: Gina Kim, Ved P. Sharma, Gargi Bandyopadhyaya, Eli Grunblatt, Sweta Roy, Nathan Agi, Binwu Tang, Esther Adler, Joan Jones, George S. Karagiannis, Xianjun Ye, David Entenberg, Sumanta Goswami, Lalage Wakefield, John S. Condeelis, Maja H. Oktay. The relationship of TMEM (tumor microenvironment of metastasis) doorways with breast cancer stem cells [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 6022.

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a β-galactoside–binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galβ1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD 100 ) that binds gal3 with picomolar affinity. TFD 100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD 100 . Because the gal3–TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD 100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.

A key step in the progression of metastatic breast cancer is the process of blood vessel intravasation. Understanding the molecular mechanisms that underlie this crucial step is essential in designing strategies to block the same with an aim to prevent metastatic disease. Recently our group has developed and published an in vitro intravasation assay (1) that recapitulates the process of intravasation and allows the use of patient derived breast cancer cells. Previously our group had identified a breast cancer invasion signature that helps facilitate clinical decision making in breast cancer patients (2). Here using fine needle aspirates from breast cancer resections we collected viable cancer cells which were labeled green using vital dyes and mixed with a macrophage cell line labeled red and layered on the top compartment of a transwell. The bottom of the transwell membrane contains a layer of extracellular matrix and a tight layer of HUVECs. Green cancer cells were separated one at a time using a florescent microscope and a micromanipulator. The cancer cells capable of intravasation are divided into two groups, one group that has crossed the entire layer and fallen to the bottom of the well, and a second group that crossed the endothelial cells but are still bound to the bottom of the endothelial layer. Cancer cells from top of the transwell which did not intravasate were also collected. RNA was extracted from the few cells collected by micromanipulation from all the three groups, amplified and converted to Cy3 labeled cDNA. The labeled cDNA was hybridized to whole genome microarrays. Gene expression was compared between the three groups of cells. Both unsupervised and supervised data analysis was performed on the three groups of cells from five patients. Significance Analysis of Microarrays, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis was performed on these datasets. Supervised analysis and validation of the altered genes was performed using qRT-PCR. Preliminary data analysis indicates the upregulation of the genes associated with Breast Cancer Stem cells and DNA repair. This is the first report of whole genome gene expression analysis from cells collected by FNA from patients, separated in vitro into intravasation competent and intravasation incompetent and unsupervised whole genome analysis performed to identify intravasation specific genes and pathways. Once unique targets and pathways are identified in intrvasation competent cells, a number of additional function blocking steps will be designed and undertaken. (1) Pignatelli J., Goswami S., et. al. Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration. Sci Signal. 2014 Nov 25;7(353) (2) Karagiannis GS., Goswami S., Jones JG., Oktay MH., and Condeelis JS. Signatures of breast cancer metastasis at a glance. J Cell Sci. 2016 May 1;129(9):1751-8. Citation Format: Gargi Bandyopadhyaya, Srinjoy Goswami, John S. Condeelis, Maja H. Oktay, Sumanta Goswami. Breast cancer intravasation signature from patient fine-needle aspirates [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 1073.

Nicotine, the well known addictive chemical of tobacco and active medication for several diseases, has proven to be a potential genotoxic compound. Although it is absorbed through lungs with smoking and mainly metabolized in liver, its effect on liver injuries is not clear. This study was designed to evaluate the genotoxicity of nicotine and corresponding the protective role of curcumin against nicotine on liver of female populations particularly who used tobacco but deprived of healthy diet. The effects were investigated by measurement of total DNA concentration of liver tissues and Comet assay of liver tissue DNA damage of female rats maintained under normal and restricted protein diets. Total DNA contents in the liver tissues were observed to decrease more significantly (P

Galectin-3, a member of a β-galactoside-binding protein family, is involved in normal growth development as well as cancer progression and metastasis, but the detailed mechanisms of its functions or its transcriptional regulations are not well understood. Besides, several regulatory elements such as GC box, CRE motif, AP-1 site, and NF-κB sites, the promoter of galectin-3 gene (LGALS3) contains several CpG islands that can be methylated during tumorigenesis of prostate leading to the gene silencing. Here we describe protocols for identification of galectin-3 DNA methylation, suppression of DNA methyltransferases to reactivate galectin-3 expression, and development of methylation-specific polymerase chain reaction (MS-PCR) to assess galectin-3 expression in various biological specimens such as tissue, serum, and urine samples.

Galectin-3, a member of a β-galactoside-binding protein family, is involved in normal growth development as well as cancer progression and metastasis, but the detailed mechanisms of its functions or its transcriptional regulations are not well understood. Besides, several regulatory elements such as GC box, CRE motif, AP-1 site, and NF-κB sites, the promoter of galectin-3 gene (LGALS3) contains several CpG islands that can be methylated during tumorigenesis of prostate leading to the gene silencing. Here we describe protocols for identification of galectin-3 DNA methylation, suppression of DNA methyltransferases to reactivate galectin-3 expression, and development of methylation-specific polymerase chain reaction (MS-PCR) to assess galectin-3 expression in various biological specimens such as tissue, serum, and urine samples.

Cell surface biomarkers CD44, CD24, CD133 and intracellular biomarker ALDH1 have been used to identify breast cancer stem cells (BCSC). BCSC are clinically significant because they are resistant to chemotherapy and in very small numbers can initiate tumor growth and metastasis. Recent studies suggest that juxtacrine signaling from monocytes and macrophages support the BCSC niche. Macrophages represent one of the components of the micro-anatomical sites of hematogenous dissemination of breast cancer cells called Tumor MicroEnvironment of Metastasis (TMEM) which are predictors of metastatic outcome in patients. We hypothesize that TMEM rich tumor microenvironments support BCSCs which are a prerequisite for systemic metastatic outgrowths. We used flow cytometry to quantify CD44high/CD24low cells, as well as mRNA fluorescent in situ hybridization (FISH) and qRT-PCR to quantify CD133 and ALDH1 expression in breast cancer cells from 50 invasive ductal carcinomas obtained from patients’ cancer excisions by fine needle aspiration (FNA) before formalin fixation. Formalin-fixed paraffin embedded tissue from each sample was also analyzed for TMEM score using triple immunohistochemistry and the stem cell marker expression was compared to TMEM scores for each corresponding cancer excision. We observed very strong correlation between the percentage of CD44high/CD24low cells and TMEM scores (r = 0.91), as well as the percentage of CD133 and ALDH1 expressing cells with TMEM scores (r = 0.88 and 0.86 respectively). FISH results were validated using qRT-PCR and showed very strong correlation with TMEM scores (r = 0.76 and 0.73 for CD133 and ALDH1 respectively). Wnt/β-catenin signaling pathway is involved in stem cell generation and pathogenesis of various types of cancer. Aberrant activation of Wnt-signaling pathway in normal stem cells can promote their transformation into cancer stem cells. We hypothesize that interactions between cancer cells and macrophages, and/ or cancer cells and endothelial cells (i.e. both interactions among the cellular components of TMEM) induce cancer stemness via Wnt/-β catenin pathway. Indeed, co-culture of mouse macrophages (BAC) with MCF-7 cells, as well as human endothelial cells (HUVEC) with MCF-7, significantly increased the percentage of cells expressing stem cells markers, while salinomycin, selective inhibitor of breast cancer stem cell growth and progression, significantly reduced BAC or HUVEC induced increase in the percentage of cancer stem cells. Our findings indicate that TMEM-rich microenvironments support breast cancer stem cells likely via Wnt signaling involving juxtacrine cancer cell-macrophage and cancer cell-endothelial cell interactions. Citation Format: Gargi Bandyopadhyaya, Eli Grunblatt, Sweta Roy, Nathan Agi, Esther Adler, Joan Jones, John S. Condeelis, Maja H. Oktay, Sumanta Goswami. Tumor microenvironment of metastasis (TMEM) in breast cancer patients represents a stem cell niche; likely mediated by the Wnt pathway. [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 3317.