Human Carcinoma-associated Fibroblasts Research Articles

The impact of stromal Hic-5 on the tumorigenesis of colorectal cancer through lysyl oxidase induction and stromal remodeling.

Carcinoma-associated fibroblasts (CAFs) influence tumor initiation, progression, and metastasis within the tumor-associated stroma. This suggests that CAFs would be a potential target for tumor therapy. Here we found that Hydrogen peroxide-inducible clone-5 (Hic-5), also named transforming growth factor beta-1-induced transcript 1 protein (Tgfb1i1), was strongly induced in CAFs found in human colorectal cancer. To investigate the role of Hic-5 in CAFs, we isolated CAFs and the control counterpart normal fibroblasts (NFs) from human colorectal cancer and non-cancerous regions, respectively. Hic-5 was highly expressed in isolated human CAFs and strongly induced in NFs in culture by the supernatant from cultured colorectal cancer cells as well as cytokines such as TGF-β, IL-1β and stromal cell-derived factor 1 (SDF-1/CXCL12). Furthermore, tumor growth was inhibited in a co-culture assay with Hic-5 knockdown fibroblasts compared with control fibroblasts. To clarify the function and significance of Hic-5 in colorectal cancer in vivo, we utilized a mouse model of azoxymethane (AOM)-induced colorectal cancer using Hic-5-deficient mice. Lack of Hic-5 in CAFs completely prevented AOM-induced colorectal cancer development in the colon tissues of mice. Mechanistic investigation revealed that Hic-5 promoted the expression of lysyl oxidase and collagen I in human control counterpart fibroblasts. Taken together, these results demonstrate that Hic-5 in CAFs is responsible for orchestrating or generating a tumor-promoting stroma.

Analysis of gene expression changes associated with human carcinoma-associated fibroblasts in non-small cell lung carcinoma

BackgroundThis study aimed to investigate the gene expression changes associated with carcinoma-associated fibroblasts (CAFs) involving in non-small cell lung carcinoma (NSCLC).MethodsWe downloaded the GEO series GSE22862, which contained matched gene expression values for 15 CAF and normal fibroblasts samples, and series GSE27289 containing SNP genotyping for four matched NSCLC samples. The differentially expressed genes in CAF samples were identified using the limma package in R. Then we performed gene ontology (GO) and pathway enrichment analysis and protein–protein interaction (PPI) network construction using the identified DEGs. Moreover, aberrant cell fraction, ploidy, allele-specific copy number, and loss of heterozygosity (LOH) within CAF cells were analyzed using the allele-specific copy number analysis.ResultsWe obtained 545 differentially expressed genes between CAF and normal fibroblasts samples. The up-regulated genes are mainly involved in GO terms such as positive regulation of cell migration and extracellular region, while the down-regulated genes participate in the lung development and extracellular region. Multiple genes including bone morphogenetic protein 4 (BMP4) and transforming growth factor, beta 3 (TGFB3) are involved in the TGF-β signaling pathway. Genes including BMP4, TGFBI and matrix Gla protein (MGP) were hub genes. Moreover, no LOH event for BMP4 and MGP was found, that for sphingosine kinase 1 (SPHK1) was 70%, and for TGFBI was 40%.ConclusionOur data suggested that BMP4, MGP, TGFBI, and SPHK1 may be important in CAFs-associated NSCLC, and the abnormal expression and high LOH frequency of them may be used as the diagnosis targets of CAFs in NSCLC.

Abstract 3587: Carcinoma-associated fibroblast-mediated regulation of anoikis in breast cancer cells

Abstract There is accumulating evidence that carcinoma-associated fibroblasts (CAFs) in the microenvironment are critical players in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (Cav -/-), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by Cav-/- and patient-derived human CAFs led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in other non-tumorigenic and breast cancer cell lines. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space. In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Additionally, we discovered that the degradation of Mcl-1 was strongly inhibited by CAF-conditioned media suggesting that Mcl-1 was not properly targeted to the proteasome. Stability of Mcl-1 is triggered by activation of ILK and p-ERK and inhibition of Gsk-3β. Furthermore, using proteomic analyses, we have identified proteins of the IGFBP family that are differentially secreted by CAFs in a fashion that is both necessary for anoikis inhibition by CAFs and sufficient to inhibit anoikis on their own. Lastly, we found that tumor formation in immunocompromised mice was reduced when cancer cells were co-injected with IGFBP-deficient CAFs rather than normal CAFs. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis. Citation Format: Zachary T. Schafer. Carcinoma-associated fibroblast-mediated regulation of anoikis in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3587. doi:10.1158/1538-7445.AM2014-3587

Abstract 1634: The regulation of anoikis by carcinoma-associated fibroblasts in breast cancer cells.

Abstract The tumor microenvironment is now appreciated to be an active participant in driving the oncogenic behavior of cancer cells. In particular, evidence is mounting that carcinoma-associated fibroblasts (CAFs) in the microenvironment are a critical player in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the importance of CAFs in promoting tumor progression and the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (KO 3T3s), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by KO 3T3s led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. We validated these findings by demonstrating that factors secreted from patient-derived human CAFs can inhibit anoikis to a similar degree. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in other non-tumorigenic cell lines and in breast cancer cell lines. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space. In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Surprisingly, this inhibition was independent of signaling through PI(3)K or MAPK and did not involve alterations in Bim protein levels. However, we discovered that the degradation of Mcl-1 was strongly inhibited by CAF-conditioned media suggesting that Mcl-1 was not properly targeted to the proteasome. Furthermore, using mass spectrometric analyses, we have identified proteins of the IGFBP family that are differentially secreted by CAFs in a fashion that regulates anoikis induction. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis. Citation Format: Kelsey Weigel, Ana Jakimenko, Brooke Conti, William Kaliney, Matthew M. Champion, Zachary T. Schafer. The regulation of anoikis by carcinoma-associated fibroblasts in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2013-1634

Abstract 2083: The regulation of anoikis by carcinoma-associated fibroblasts in breast cancer cells

Abstract The initial step of the metastatic cascade in most carcinomas occurs when tumor cells break through the basement membrane and invade locally into the surrounding microenvironment. This microenvironment is now appreciated to be an active participant in driving the oncogenic behavior of cancer cells. In particular, evidence is mounting that carcinoma-associated fibroblasts (CAFs) in the microenvironment are a critical player in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the importance of CAFs in promoting tumor progression and the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (KO 3T3s), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by KO 3T3s led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. We validated these findings by demonstrating that factors secreted from patient-derived human CAFs can inhibit anoikis to a similar degree. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in two additional non-tumorigenic cell lines: BPH-1 and HMEC. In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Surprisingly, this inhibition was independent of signaling through PI(3)K or MAPK and did not involve alterations in Bim protein levels. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2083. doi:1538-7445.AM2012-2083

Abstract 48: Activation of c-Met receptor signaling by carcinoma-associated fibroblasts in the tumor microenvironment

Abstract Objective: Carcinoma-associated fibroblasts (CAFs) play a key role in tumorigenesis by promoting tumor growth, invasion, and metastasis. At sites of intra-abdominal ovarian cancer metastases, more than half of the tumor microenvironment is composed of CAFs, however, the mechanism for how CAFs promote metastatic disease in ovarian cancer is not known. Because of the prominent role that the receptor tyrosine kinase c-Met plays in metastasis, we hypothesized that CAFs within the metastatic tumor microenvironment may activate c-Met signaling in ovarian cancer cells leading to tumor progression at a metastatic site. Methods: Human cancer cell lines and primary human carcinoma-associated fibroblasts isolated from patients’ ovarian cancer metastases were used in multiple biochemical assays to analyze the role of CAFs in c-Met receptor signaling. Results: Using a receptor tyrosine kinase array to analyze phosphorylation of 42 different receptor tyrosine kinases (RTKs), we found that c-Met was the only RTK that was phosphorylated in cancer cells upon co-culture with CAFs. Further analysis of the co-culture revealed that direct contact between cancer cells and CAFs was required for activation of c-Met. Interestingly, the unique ligand for c-Met, hepatocyte growth factor (HGF), could not be detected in conditioned media from CAFs by ELISA and addition of HGF neutralizing antibodies to the co-culture did not prevent activation of the receptor. Furthermore, CAFs transiently transfected with HGF siRNA were still able to activate c-Met in the human ovarian cancer cell line SKOV3ip1 suggesting that c-Met activation by CAFs is HGF-independent. This effect was not limited to ovarian cancer cell lines as co-culture of CAFs with the cervical cancer cell line, HeLa, which expresses wild-type c-Met, and the breast cancer cell line, T47D, showed the same results as the two ovarian cancer cell lines, SKOV3ip1 and OVCAR-5. Remarkably, membrane fragments isolated from CAFs were sufficient to stimulate phosphorylation of c-Met in cancer cells in an HGF-independent manner. Several candidate factors were identified in the CAF membrane proteome which can activate c-Met signaling. Conclusions: Collectively, these results suggest a potentially novel mechanism whereby CAFs activate c-Met in cancer cells through direct contact which provides an additional rationale for the therapeutic targeting of c-Met in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 48. doi:1538-7445.AM2012-48

Breast Carcinoma–Associated Fibroblasts Rarely Contain p53 Mutations or Chromosomal Aberrations

It has become increasingly clear that the cells within the tumor microenvironment play a critical role in cancer growth and metastasis. Studies in experimental models suggest that carcinoma-associated fibroblasts (CAF) differ from normal fibroblasts and are capable of promoting cancer progression through a variety of mechanisms. At present, a definitive view is lacking on whether genomic abnormalities are present and whether they might underlie the observed phenotypic differences. This study reports the molecular analysis of the largest series of breast CAFs reported to date, with an array comparative genomic hybridization-based DNA copy number analysis of cultured CAFs derived from 25 freshly resected human breast cancers. We found DNA copy number changes consisting of the whole arm of chromosomes 6p and 9p plus interstitial 4q loss in only one sample. No abnormalities were observed in non-tumor-associated fibroblast counterparts. Karyotyping of the same CAF revealed further chromosomal abnormalities, which included clonal loss of chromosomes, chromosomal duplications, and less frequent chromosomal rearrangements. These abnormalities were not associated with alterations in the global gene expression profile of this particular CAF, relative to its non-tumor-associated fibroblast counterpart. Moreover, this particular patient's CAF also displayed the only p53 mutation in the cohort, the first time such a mutation has been reported in freshly cultured human CAFs. These findings argue that the procancerous effects of CAFs are unlikely to be due to DNA copy number-type genomic abnormalities in the CAFs themselves. As such, breast CAFs should be mainly regarded as genomically stable cellular constituents that exist within complex cancer microenvironments.

Abstract 1015: Head and neck cancer expression of YAP65: A novel oncogene

Abstract Yes-associated protein (YAP), a transcription coactivator associated with maintaining tissue size, is reported as an oncogene in many types of cancer including ovarian, liver, lung, colon and prostate. However, the role of YAP in head and neck carcinomas is not known. Our objective was to explore the role of YAP in head and neck carcinomas. We found YAP overexpressed in the epithelial and stromal compartments of head and neck carcinomas compared to benign tissues in 140 samples. Interestingly, the squamous cell carcinomas, had elevated YAP expression in the epithelial cytoplasm in 36% of the patients. However, we observed both cytoplasmic and prominently nuclear localization of YAP in the adenocarcinomas. The tumor microenvironment is widely accepted as a promoter of tumor progression, where carcinoma-associated fibroblasts (CAFs) constitute the majority of cells. In the stroma, YAP was found to be expressed in endothelial cells and some heterogenously within the CAF population. As reported for prostate cancer, we found the transforming growth factor-beta (TGF-ß) receptor type II expression to be lost in the head and neck cancer-associated stromal cells. Since YAP has never been reported to be associated with CAFs, we performed in vitro experiment to determine the relationship of YAP and TGF-ß signaling in primary stromal cells associated with benign and head and neck cancer cells. We found TGF-ß1 can promote YAP expression and nuclear localization in normal fibroblasts. The oral human CAFs have differential expression of YAP and α-smooth muscle actin (α-SMA). Although CAFs are commonly characterized by their expression of α-SMA, YAP-expressing CAFs expressed less α-SMA and α-SMA-expressing CAFs expressed less YAP in seven independent CAF lines developed. However, all of these heterougenous head and neck CAF lines had the loss of TGF-ß receptor type II expression and concomitant upregulation of Wnt5a. Interestingly, those CAFs expressing YAP had 2-fold greater Wnt5a expression. In conclusion YAP expression is differentially expressed in adeno- and squamous cell carcinomas of head and neck carcinomas, and is heterogenously expressed in the associated CAF. YAP may regulate Wnt5a as a paracrine factor, reported to promote the progression of oral carcinomas, in a TGF-ß dependent and independent manner. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1015.

Immortalization of Human Carcinoma-Associated Fibroblasts from Primary Colorectal Carcinomas

Introduction: Reciprocal interplay bewteen epithelial and stromal cells contributes to the initiation, progression and metastasis of colorectal cancer. The importance of carcinoma-associated fibroblasts (CAFs) in tumor develoment is highlighted by the observation that nontumorigenic prostate epithelial cells can form tumors in nude mice when co-implanted with CAF, but not when co-implanted with fibroblasts from benign prostatic hypertrophy. The precise role of colorectal CAFs in tumor-stroma interactions has been difficult to study, in part because of the difficulty of obtaining primary CAFs in sufficient quantity for experiments. Once isolated, the CAFs can be passaged for a limited number of times before they undergo senescence. Further characterization of human colorectal CAFs would provide the basis for stromal-specific therapeutic targeting in colorectal cancers. The purpose of this study was two-fold: 1) immortalize human CAFs derived from colorectal cancers in order to begin to investigate their influence on human colorectal cancer cells and 2) document gene-specific changes attributable to the immortalization process. Methods: Freshly dissected and dissociated cells were cultured from a resected colon cancer specimen under an IRB-approved protocol. The patient had not received prior chemoradiotherapy. The cells were cultured on collagen I and the purity of the fibroblast culture was verified by immunohistochemistry. Fibroblasts are positive for vimentin and/or α-smooth muscle actin, but negative for cytokeratin. CAFs were infected with lentiviral vectors containing a CMV promotor upstream of SV40 Large T antigen and human telomerase. Total RNA was extracted using RNAqueous (Ambion) from triplicate cultures of transformed (t)CAFs and parental (p)CAFs. The purity and concentration of the RNA samples were determined using Agilent 2100 Bioanalyzer. Samples were hybridized to Affymetrix Gene Chips (HGU133 Plus 2.0). Analyses using the Partek Genomics Suite included routine quality controls, raw data background subtraction and normalization. Differentially expressed candidate genes were identified using the Benjamani-Hochberg multiple hypothesis comparisons (ANOVA, p<0.01 and p<0.005) to decrease the false discovery rate. Ingenuity Pathways analyses were performed to identify differences between the t- and p-CAFs. Results: ANOVA identified 2779 and 648 genes that were significant at the p≤0.01 and 0.005 level, respectively. The major pathways representing the genes differentially expressed bewteen the t- and p-CAFs include: 1) the transcriptional regulator TP53 2) growth factor TGFB1, 3) mitogen-activated protein kinase pathways (p38 and p-extracellularly regulated kinase), and 4) the transcriptional regulator nuclear factor-κB. As expected, one canonical pathway that was altered by the transformation, were genes involved in cell cycle and G1/S checkpoint regulation. Conclusions: The immortalization and subsequent careful characterization of human colorectal CAFs provides an invaluable tool for further study of stromal-epithelial interactions in colorectal carcinogenesis and metastasis. Although genes related to the cell cycle has been significantly altered, future studies are required to verify that the t-CAFs alone are non-tumorigenic in nude mice, similar to the p-CAFs. Additionally, immortalized normal fibroblasts derived from the normal a colonic mucosa will need to be generated as an important control.