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Breast Cancer Anti-Estrogen Resistance 3 (BCAR3) in Colorectal Cancer Migration and Intestinal Homeostasis
Su1786
AGA Abstracts
UPREGULATED CLAUDIN-2 EXPRESSION IN INTESTINAL EPITHELIUM AUGMENTS SUSCEPTIBILITY TO APC-MEDIATED COLON CANCER GROWTH AND MALIGNANCY Rizwan Ahmad, Balawant Kumar, Punita Dhawan, Amar B. Singh Among claudin proteins expressed in the colon, claudin-2 is unique as its expression is restricted to the undifferentiated and proliferative crypt base. Also, claudin-2 expression is highly upregulated in spontaneous colon cancer (CRC) and colitis-associated cancer (CAC). Moreover, forced claudin-2 expression in claudin-2 deficient CRC cells promoted their tumorigenic ability in mouse xenograft assay. In vivo, claudin-2 overexpression in intestinal epithelial cells (Cldn-2TG mice) induced a longer, leakier and hyper-proliferative colon. However, despite the leaky gut, Cldn-2TG mice exhibited immune suppressive phenotype. Considering the key role of inflammation in colon tumorigenesis, we therefore examined how claudin-2 expression may affect colon tumorigenesis under conditions of APC mutation, key regulator of colon carcinogenesis. Inter-breeding of Cldn-2TG and APCmin mice generated APCmin/Cldn2 mice which demonstrated significant increases in intestinal tumor burden (248+40%, p<0.001) and colonic tumor burden [Incidence (172.50±18%, p<0.001) and size (521.5±34% vs APCmin, p<0.001)] compared to APCmin mice. H&E analysis further affirmed dedifferentiated phenotype of APCmin/Cldn2 mice tumors (vs APCmin mice tumors). APCmin/Cldn2 mice also survived less than APCmin mice. Immunoblot analysis demonstrated marked suppression of Stat-3 and NF-kB signaling in APCmin/Cldn2 mice (vs APCmin mice) suggesting immune suppressive environment. High throughput transcriptome analysis revealed significant upregulation of CTNNB1 and tumor promotive genes including c-myc (3.425 ± 0.71-fold, p<0.05), cyclin D1 (8.203 ± 2.5-fold, p<0.05), Ki67 (11.04 ± 2.4-fold, p<0.05) and TGF-β (3.591 ± 0.32-fold, p<0.01) in APCmin/Cldn-2 mice (vs APCmin mice). Similar increases in cancer stem cell markers LGR5 (7.110 ± 1.7-fold, p<0.05), OLFM4 (4.386 ± 0.47-fold, p<0.05) and CD133 (3.005 ± 0.69-fold, p<0.05) were documented. Continued investigation using CRC cells and in vitro model of spontaneous differentiation demonstrated inverse association between differentiation and claudin-2 expression. Accordingly, forced differentiation using clinically pertinent HDAC-inhibitor (HDACi), SAHA (Suberoylanilide Hydroxamic Acid), significantly suppressed claudin-2 protein expression and promoter activity (4.59 ± 0.53% vs con, p<0.0001 and 8.425 ± 0.78% vs con, p>0.001) in CRC cells. Accompanying upregulation of P27/KIP1, a cell cycle inhibitor and marker of differentiated cells (393.3 ± 61.18% vs con, p>0.05) supported validity of the model. Taken together, our data supports key role of claudin-2 expression in promoting colon cancer malignancy by modulating epithelial homeostasis and colon microenvironment. We further identify epigenetic regulation as mechanism underlying claudin-2 regulation in colon cancer which can be harnessed for therapeutic advantages.
Su1785 BREAST CANCER ANTI-ESTROGEN RESISTANCE 3 (BCAR3) IN COLORECTAL CANCER MIGRATION AND INTESTINAL HOMEOSTASIS Joshua J. Thompson, Kan He, Sarah P. Short, Xi Chen, Cody Keating, Yash A. Choksi, Christopher S. Williams Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-to-mesenchymal transition (EMT) and functions as a tumor suppressor in colorectal cancer (CRC). Through a yeast two-hybrid screen, BVES was shown to interact with breast cancer anti-estrogen resistance 3 (BCAR3). BCAR3 was originally identified via its upregulation in tamoxifen resistant breast cancer cell lines and functions as an adapter protein, recruiting p130Cas to focal adhesions. We first sought to define the role of BCAR3 in CRC to aid in determining the functional consequence of the BVES-BCAR3 interaction. We began by analyzing the combined Moffitt Cancer Center/Vanderbilt University Medical Center CRC expression array data set consisting of normal, adenomatous, and 250 tumor samples organized by tumor stage. BCAR3 was downregulated in adenomas (p = 0.01075) and further decreased at all CRC stages (p = 4.527e-07). Analysis of TCGA RNASeq data confirmed reductions at all stages (p < 2.2e-16). BCAR3 expression by qRT-PCR was also reduced by three-fold (p < 0.001) in two out of three paired normal and tumor samples acquired from the Cooperative Human Tissue Network (CHTN). Not surprisingly, heterogeneity also existed in BCAR3 protein levels in a panel of CRC cell lines. In Caco2 cells (which express low levels of BCAR3), stable overexpression of BCAR3 impaired migration through uncoated transwells, and reduced both cellular proliferation, as measured by cell number, and clonogenic growth capacity. Stable lentiviral expression of BCAR3 in Caco2 cells impaired migration on uncoated substrates (33.9 ± 1.12 vs. 51.0 ± 4.1 percent wound closure at 48 hours, p = 0.0003) but not on collagen coated or fibronectin coated surfaces as assayed using the magnetically attachable stencils (MAts) assay. shRNA knockdown of BCAR3 and CrisprCas9 mediated knockout of BCAR3 in HCT116 cells impaired transwell migration but did not alter proliferation. Additionally, intestinal enteroid 3D cultures from Bcar3-/- mice had increased branching (4.17 ± 0.32 vs. 2.90 ± 0.33 average branches per day five enteroid, p < 0.01) and have at least 1.4-fold increases in expression of the Wnt target genes Lgr5, Axin2, and Myc (p < 0.01), suggestive of perturbations in the crypt/stem cell compartments. Together, these results implicate BCAR3 in stem cell dynamics, cell migration, and in overall epithelial homeostasis; all processes relevant in the pathogenesis of CRC.
Su1787 EXOSOMES DERIVED FROM COLON CANCER CELLS PROMOTE TUMOR PROGRESSION VIA THE ACTIVATION OF FIBROBLASTS IN TUMOR MICROENVIRONMENT Yoshito Hayashi, Shunsuke Yoshii, Yoshiki Tsujii, Takanori Inoue, Keiichi Kimura, Akihiko Sakatani, Kengo Nagai, Tetsuji Fujinaga, Satoshi Hiyama, Takahiro Inoue, Shinichiro Shinzaki, Kenji Watabe, Hideki Iijima, Tetsuo Takehara Background and Aim: Tumor microenvironment offers favorable conditions for the tumor progression, invasion and metastasis. Activation of fibroblasts surrounding cancer cells known as cancer associated fibroblasts (CAFs) plays a pivotal role. However, the mechanism of crosstalk between CAFs and cancer cells remains unclear. We aimed to reveal the role of cancer-derived exosomes (CDEs) as the communication tool between cancer cells and fibroblasts. Methods: We evaluated the effects of exosomes on tumor growth associated with the interaction between cancer cells and fibroblasts in vitro and in vivo, using human cell lines (p53 wild colon cancer; HCT116, p53 mutant colon cancer; HT29 and fibroblast; CCD-18Co and WI-38) and xenograft model with immune-deficient nude mice. CDEs were isolated from conditioned media of cancer cells using sequential ultracentrifugation method. Fluorescent microscopy was used to determine the uptake of CDEs labelled with PKH67 dye by fibroblasts. The expressions of alpha-smooth muscle actin (αSMA), transforming growth factor beta 1 (TGFβ1) and vascular endothelial growth factor A (VEGFA) mRNA in fibroblasts were analyzed by real time qRT-PCR. The expressions of p53 and fibroblast activation markers were also analyzed in human colon cancer tissues immunohistochemically. Results: CDEs were confirmed to be isolated by the expression of CD9 and CD63. CCD18Co and WI-38 fibroblasts demonstrated time-dependent uptake of CDEs. Incubation of fibroblasts with CDEs increased the expression of αSMA, TGFβ1 and VEGFA in vitro. HT29 cells subjected with knock-down of Rab27a, which is indispensable for the excretion of exosome, or control cells with/without fibroblasts were subcutaneously injected to nude mice. Control HT29 cells showed accelerated tumor growth in the presence of fibroblasts. In contrast, tumor growth was significantly reduced by suppression of Rab27a in HT29 cells not in the absence but in the presence of fibroblasts. Additionally, p53-wild-type HCT116 cells co-injected with fibroblasts did not show increased tumor growth in contrast to the accelerated tumor growth in p53-mutant HT29 cells or p53-deficient HCT116 cells. The accelerated tumor progression of p53-deficient HCT116 cells with fibroblasts was also cancelled by suppression of CDEs secretion using knock-down of Rab27a. Microarray analysis revealed that exosomes derived from p53-suppressed cancer cells showed different profile of microRNA from p53-wild cells. Immunohistochemistry of human colon cancer tissues demonstrated the positive correlations between p53 staining of cancer cells and αSMA and VEGF staining in the stromal area. Conclusion: Our results suggest that CDEs play a pivotal role in tumor progression via the activation of fibroblasts associated with the loss or mutant of p53 of cancer cells. CDEs might be a novel therapeutic target for colorectal cancers.
Figure 1 | Loss of BCAR3 increases enteroids branching and expression of WNT targets. WT and Bcar3-/- small intestine crypts were isolated from 8-week old mice and plated in 3D matrigel cultures. (A) Bcar3-/- enteroids have increased branches at Day 3, Day 4, and Day 5 post plating. (B) Day 5 Bcar3-/- enteroids branches are larger and exhibit more spheroidal morphologically than WT enteroids. Arrows indicate quantified branches in each image. (C) Expression of Wnt targets Lgr5, Axin2, and Myc is increased in Bcar3-/- enteroids. * p<0.05, ** p<0.01, *** p<0.001.
AGA Abstracts
S-558
AGA Abstracts
UPREGULATED CLAUDIN-2 EXPRESSION IN INTESTINAL EPITHELIUM AUGMENTS SUSCEPTIBILITY TO APC-MEDIATED COLON CANCER GROWTH AND MALIGNANCY Rizwan Ahmad, Balawant Kumar, Punita Dhawan, Amar B. Singh Among claudin proteins expressed in the colon, claudin-2 is unique as its expression is restricted to the undifferentiated and proliferative crypt base. Also, claudin-2 expression is highly upregulated in spontaneous colon cancer (CRC) and colitis-associated cancer (CAC). Moreover, forced claudin-2 expression in claudin-2 deficient CRC cells promoted their tumorigenic ability in mouse xenograft assay. In vivo, claudin-2 overexpression in intestinal epithelial cells (Cldn-2TG mice) induced a longer, leakier and hyper-proliferative colon. However, despite the leaky gut, Cldn-2TG mice exhibited immune suppressive phenotype. Considering the key role of inflammation in colon tumorigenesis, we therefore examined how claudin-2 expression may affect colon tumorigenesis under conditions of APC mutation, key regulator of colon carcinogenesis. Inter-breeding of Cldn-2TG and APCmin mice generated APCmin/Cldn2 mice which demonstrated significant increases in intestinal tumor burden (248+40%, p<0.001) and colonic tumor burden [Incidence (172.50±18%, p<0.001) and size (521.5±34% vs APCmin, p<0.001)] compared to APCmin mice. H&E analysis further affirmed dedifferentiated phenotype of APCmin/Cldn2 mice tumors (vs APCmin mice tumors). APCmin/Cldn2 mice also survived less than APCmin mice. Immunoblot analysis demonstrated marked suppression of Stat-3 and NF-kB signaling in APCmin/Cldn2 mice (vs APCmin mice) suggesting immune suppressive environment. High throughput transcriptome analysis revealed significant upregulation of CTNNB1 and tumor promotive genes including c-myc (3.425 ± 0.71-fold, p<0.05), cyclin D1 (8.203 ± 2.5-fold, p<0.05), Ki67 (11.04 ± 2.4-fold, p<0.05) and TGF-β (3.591 ± 0.32-fold, p<0.01) in APCmin/Cldn-2 mice (vs APCmin mice). Similar increases in cancer stem cell markers LGR5 (7.110 ± 1.7-fold, p<0.05), OLFM4 (4.386 ± 0.47-fold, p<0.05) and CD133 (3.005 ± 0.69-fold, p<0.05) were documented. Continued investigation using CRC cells and in vitro model of spontaneous differentiation demonstrated inverse association between differentiation and claudin-2 expression. Accordingly, forced differentiation using clinically pertinent HDAC-inhibitor (HDACi), SAHA (Suberoylanilide Hydroxamic Acid), significantly suppressed claudin-2 protein expression and promoter activity (4.59 ± 0.53% vs con, p<0.0001 and 8.425 ± 0.78% vs con, p>0.001) in CRC cells. Accompanying upregulation of P27/KIP1, a cell cycle inhibitor and marker of differentiated cells (393.3 ± 61.18% vs con, p>0.05) supported validity of the model. Taken together, our data supports key role of claudin-2 expression in promoting colon cancer malignancy by modulating epithelial homeostasis and colon microenvironment. We further identify epigenetic regulation as mechanism underlying claudin-2 regulation in colon cancer which can be harnessed for therapeutic advantages.
Su1785 BREAST CANCER ANTI-ESTROGEN RESISTANCE 3 (BCAR3) IN COLORECTAL CANCER MIGRATION AND INTESTINAL HOMEOSTASIS Joshua J. Thompson, Kan He, Sarah P. Short, Xi Chen, Cody Keating, Yash A. Choksi, Christopher S. Williams Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-to-mesenchymal transition (EMT) and functions as a tumor suppressor in colorectal cancer (CRC). Through a yeast two-hybrid screen, BVES was shown to interact with breast cancer anti-estrogen resistance 3 (BCAR3). BCAR3 was originally identified via its upregulation in tamoxifen resistant breast cancer cell lines and functions as an adapter protein, recruiting p130Cas to focal adhesions. We first sought to define the role of BCAR3 in CRC to aid in determining the functional consequence of the BVES-BCAR3 interaction. We began by analyzing the combined Moffitt Cancer Center/Vanderbilt University Medical Center CRC expression array data set consisting of normal, adenomatous, and 250 tumor samples organized by tumor stage. BCAR3 was downregulated in adenomas (p = 0.01075) and further decreased at all CRC stages (p = 4.527e-07). Analysis of TCGA RNASeq data confirmed reductions at all stages (p < 2.2e-16). BCAR3 expression by qRT-PCR was also reduced by three-fold (p < 0.001) in two out of three paired normal and tumor samples acquired from the Cooperative Human Tissue Network (CHTN). Not surprisingly, heterogeneity also existed in BCAR3 protein levels in a panel of CRC cell lines. In Caco2 cells (which express low levels of BCAR3), stable overexpression of BCAR3 impaired migration through uncoated transwells, and reduced both cellular proliferation, as measured by cell number, and clonogenic growth capacity. Stable lentiviral expression of BCAR3 in Caco2 cells impaired migration on uncoated substrates (33.9 ± 1.12 vs. 51.0 ± 4.1 percent wound closure at 48 hours, p = 0.0003) but not on collagen coated or fibronectin coated surfaces as assayed using the magnetically attachable stencils (MAts) assay. shRNA knockdown of BCAR3 and CrisprCas9 mediated knockout of BCAR3 in HCT116 cells impaired transwell migration but did not alter proliferation. Additionally, intestinal enteroid 3D cultures from Bcar3-/- mice had increased branching (4.17 ± 0.32 vs. 2.90 ± 0.33 average branches per day five enteroid, p < 0.01) and have at least 1.4-fold increases in expression of the Wnt target genes Lgr5, Axin2, and Myc (p < 0.01), suggestive of perturbations in the crypt/stem cell compartments. Together, these results implicate BCAR3 in stem cell dynamics, cell migration, and in overall epithelial homeostasis; all processes relevant in the pathogenesis of CRC.
Su1787 EXOSOMES DERIVED FROM COLON CANCER CELLS PROMOTE TUMOR PROGRESSION VIA THE ACTIVATION OF FIBROBLASTS IN TUMOR MICROENVIRONMENT Yoshito Hayashi, Shunsuke Yoshii, Yoshiki Tsujii, Takanori Inoue, Keiichi Kimura, Akihiko Sakatani, Kengo Nagai, Tetsuji Fujinaga, Satoshi Hiyama, Takahiro Inoue, Shinichiro Shinzaki, Kenji Watabe, Hideki Iijima, Tetsuo Takehara Background and Aim: Tumor microenvironment offers favorable conditions for the tumor progression, invasion and metastasis. Activation of fibroblasts surrounding cancer cells known as cancer associated fibroblasts (CAFs) plays a pivotal role. However, the mechanism of crosstalk between CAFs and cancer cells remains unclear. We aimed to reveal the role of cancer-derived exosomes (CDEs) as the communication tool between cancer cells and fibroblasts. Methods: We evaluated the effects of exosomes on tumor growth associated with the interaction between cancer cells and fibroblasts in vitro and in vivo, using human cell lines (p53 wild colon cancer; HCT116, p53 mutant colon cancer; HT29 and fibroblast; CCD-18Co and WI-38) and xenograft model with immune-deficient nude mice. CDEs were isolated from conditioned media of cancer cells using sequential ultracentrifugation method. Fluorescent microscopy was used to determine the uptake of CDEs labelled with PKH67 dye by fibroblasts. The expressions of alpha-smooth muscle actin (αSMA), transforming growth factor beta 1 (TGFβ1) and vascular endothelial growth factor A (VEGFA) mRNA in fibroblasts were analyzed by real time qRT-PCR. The expressions of p53 and fibroblast activation markers were also analyzed in human colon cancer tissues immunohistochemically. Results: CDEs were confirmed to be isolated by the expression of CD9 and CD63. CCD18Co and WI-38 fibroblasts demonstrated time-dependent uptake of CDEs. Incubation of fibroblasts with CDEs increased the expression of αSMA, TGFβ1 and VEGFA in vitro. HT29 cells subjected with knock-down of Rab27a, which is indispensable for the excretion of exosome, or control cells with/without fibroblasts were subcutaneously injected to nude mice. Control HT29 cells showed accelerated tumor growth in the presence of fibroblasts. In contrast, tumor growth was significantly reduced by suppression of Rab27a in HT29 cells not in the absence but in the presence of fibroblasts. Additionally, p53-wild-type HCT116 cells co-injected with fibroblasts did not show increased tumor growth in contrast to the accelerated tumor growth in p53-mutant HT29 cells or p53-deficient HCT116 cells. The accelerated tumor progression of p53-deficient HCT116 cells with fibroblasts was also cancelled by suppression of CDEs secretion using knock-down of Rab27a. Microarray analysis revealed that exosomes derived from p53-suppressed cancer cells showed different profile of microRNA from p53-wild cells. Immunohistochemistry of human colon cancer tissues demonstrated the positive correlations between p53 staining of cancer cells and αSMA and VEGF staining in the stromal area. Conclusion: Our results suggest that CDEs play a pivotal role in tumor progression via the activation of fibroblasts associated with the loss or mutant of p53 of cancer cells. CDEs might be a novel therapeutic target for colorectal cancers.
Figure 1 | Loss of BCAR3 increases enteroids branching and expression of WNT targets. WT and Bcar3-/- small intestine crypts were isolated from 8-week old mice and plated in 3D matrigel cultures. (A) Bcar3-/- enteroids have increased branches at Day 3, Day 4, and Day 5 post plating. (B) Day 5 Bcar3-/- enteroids branches are larger and exhibit more spheroidal morphologically than WT enteroids. Arrows indicate quantified branches in each image. (C) Expression of Wnt targets Lgr5, Axin2, and Myc is increased in Bcar3-/- enteroids. * p<0.05, ** p<0.01, *** p<0.001.
AGA Abstracts
S-558