Elevated expression of TGF-beta1 in head and neck cancer-associated fibroblasts

Head and neck cancers are characterized by a vigorous desmoplastic response, but the contribution of stromal-derived growth factors to the tumor microenvironment is poorly understood. We evaluated the expression of stromal growth factor expression in head and neck squamous cell carcinoma (HNSCC) in normal and tumor-associated stromal cells. Stromal tissue was isolated from epithelial cells with laser capture microdissection (LCMD) and analyzed by cDNA array for the expression of TGFalpha, TGF-beta1, HGF, PDGF-alpha, IGFII, bFGF, aFGF, VEGFC, and VEGF. Primary fibroblasts were isolated in vitro from HNSCC tumors, adjacent histologically normal mucosa, and skin in vitro. Fibroblast populations were assessed for TGF-beta1 expression by ELISA and luciferase reporter assay to assess protein expression. We identified TGF-beta1 and IGFII overexpression in normal and tumor-associated stromal cells; however, only TGF-beta1 was significantly overexpressed (3.4-fold) in tumor-associated stroma. Assessment of carcinoma-associated fibroblasts (CAFs), normal dermal fibroblasts (NDFs), and normal mucosal fibroblasts (NMFs) in propagated fibroblasts demonstrated persistently elevated levels of TGF-beta1 in CAFs compared to NMF and NDF populations. Elevated levels of TGF-beta1 were identified in the stromal compartment of HNSCC tumors compared to normal mucosa by immunohistochemical analysis. These results suggest that TGF-beta1 mRNA and protein is specifically upregulated in CAFs in vitro and in vivo.     

c-Ski activates cancer-associated fibroblasts to regulate breast cancer cell invasion

Aberrant expression of c-Ski oncoprotein in some tumor cells has been shown to be associated with cancer development. However, the role of c-Ski in cancer-associated fibroblasts (CAFs) of tumor microenvironment has not been characterized. In the current study, we found that c-Ski is highly expressed in CAFs derived from breast carcinoma microenvironment and this CAF-associated c-Ski expression is associated with invasion and metastasis of human breast tumors. We showed that c-Ski overexpression in immortalized breast normal fibroblasts (NFs) induces conversion to breast CAFs by repressing p53 and thereby upregulating SDF-1 in NFs. SDF-1 treatment or p53 knockdown in NFs had similar effects on the activation of NFs as c-Ski overexpression. The c-Ski-activated CAFs show increased proliferation, migration, invasion and contraction compared with NFs. Furthermore, c-Ski-activated CAFs facilitated the migration and invasion of MDA-MB-231 breast cancer cells. Our data suggest that c-Ski is an important regulator in the activation of CAFs and may serve as a potential therapeutic target to block breast cancer progression.     

PRL-3 initiates tumor angiogenesis by recruiting endothelial cells in vitro and in vivo

We show here that PRL-3 protein is expressed in fetal heart, developing blood vessels, and pre-erythrocytes but not in their mature counterparts. These observations imply that PRL-3 may be involved in the early development of the circulatory system. Because PRL-3 mRNA had been reported to be consistently elevated in metastatic samples derived from colorectal cancers, we attempted to investigate if PRL-3 might be involved in tumor angiogenesis and if PRL-3-expressing cells could cross-talk to human umbilical vascular endothelial cells (HUVEC) by using an in vitro coculture system. HUVECs were grown with fibroblasts, which were later overlaid with PRL-3-expressing cells. We observed that both PRL-3-expressing Chinese hamster ovary (CHO) cells and PRL-3-expressing DLD-1 human colon cancer cells could redirect the migration of HUVECs toward them; in addition, PRL-3-expressing DLD-1 cells could enhance HUVEC vascular formation. In vivo injection of PRL-3-expressing CHO cells into nude mice to form local tumors resulted in the recruitment of host endothelial cells into the tumors and initiation of angiogenesis. We further showed that PRL-3-expressing cells reduced interleukin-4 (IL-4) expression levels and thus attenuated IL-4 inhibitory effects on the HUVEC vasculature. Our findings provide direct evidence that PRL-3 may be involved in triggering angiogenesis and establishing microvasculature and it may serve as an attractive therapeutic target with respect to both angiogenesis and cancer metastasis.     

Paracrine recruitment and activation of fibroblasts by c-Myc expressing breast epithelial cells through the IGFs/IGF-1R axis

Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast-like, protumorigenic cells referred to as Cancer-Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI-38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c-Myc oncogene (MCF10A-MycER). After c-Myc activation, the conditioned medium (CM) of MCF10A-MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin-like growth factor binding protein-6 (IGFBP-6) and increased insulin-like growth factor-1 and IGF-II (IGF-I, IGF-II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP-6 or IGF-I or IGF-II expression in epithelial cells or blocking Insulin-like growth factor 1 receptor (IGF-1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI-38 fibroblasts to CM from induced MCF10A-MycER cells or to IGF-II upregulated FAK phosphorylation on Tyr³⁹⁷, as well as the expression of α-smooth muscle actin (α-SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three-dimensional (3D)-organotypic assays, WI-38 or human primary fibroblasts, preactivated with either CM from MCF10A-MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF-1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF-1R axis, which directly promotes TME remodeling and increases tumor invasion.     

Cancer associated fibroblasts stimulated by transforming growth factor beta1 (TGF-β1) increase invasion rate of tumor cells: a population study

Cancer associated fibroblasts (CAFs) are believed to promote tumor growth and progression. Our objective was to measure the effect of TGF-beta1 on fibroblasts isolated from invasive breast cancer patients. Fibroblasts were isolated from tissue obtained at surgery from patients with invasive breast cancer (CAF; n = 28) or normal reduction mammoplasty patients (normal; n = 10). Myofibroblast activation was measured by counting cells immunostained for smooth muscle alpha actin (ACTA2) in cultures +/- TGF-beta 1. Conditioned media (CM) was collected for invasion assays and RNA was isolated from cultures incubated in media +/- TGF-beta1 for 24 h. Q-PCR was used to measure expression of cyclin D1, fibronectin, laminin, collagen I, urokinase, stromelysin-1, and ACTA2 genes. Invasion rate was measured in chambers plated with MDA-MB-231 cells and exposed to CM in the bottom chamber; the number of cells that invaded into the bottom chamber was counted. Wilcox Rank Sum tests were used to evaluate differences in CAFs and normal fibroblasts and the effect of TGF-beta 1. There was no difference in percent myofibroblasts or invasion rate between normal and CAF cultures. However, TGF-beta1 significantly increased the percent of myofibroblasts (P < 0.01) and invasion rate (P = 0.02) in CAF cultures. Stromelysin-1 expression was significantly higher in normal versus CAF cultures (P < 0.01). TGF-beta 1 significantly increased ACTA2 expression in both normal and CAF cultures (P < 0.01). Expression of fibronectin and laminin was significantly increased by TGF-beta in CAF cultures (P < 0.01). CAFs were measurably different from normal fibroblasts in response to TGF-beta 1, suggesting that TGF-beta stimulates changes in CAFs that foster tumor invasion.     

PRL-3 expression in metastatic cancers.

PURPOSE: Expression of the PRL-3 tyrosine phosphatase is elevated in liver metastases derived from colorectal cancer (CRC). We sought to determine the cellular basis of this elevation and assess the expression of PRL-3 in metastatic lesions derived from cancers of the colon and other tissues. EXPERIMENTAL DESIGN: We developed modifications of in situ hybridization methods that facilitated the study of paraffin-embedded sections. We also evaluated PRL-3 gene copy numbers using fluorescence in situ hybridization and developed antibodies to assess PRL-3 subcellular localization. RESULTS: PRL-3 mRNA expression was elevated in nearly all metastatic lesions derived from CRCs, regardless of the site of metastasis (liver, lung, brain, or ovary). Expression was found in neoplastic cells, although tumor endothelium also expressed the gene. In contrast, little or no PRL-3 expression was observed in normal colon, nonmetastatic primary cancers, or metastatic lesions derived from cancers other than those of the colon (pancreas, stomach, or esophagus). Interphase fluorescence in situ hybridization confirmed that gene amplification was not the major cause of PRL-3 overexpression. Immunohistochemical analysis with anti-PRL-3 antibodies showed a cell membrane localization, consistent with the predicted isoprenylation of the protein. CONCLUSIONS: These studies establish an unexpected and unprecedented specificity in metastatic gene expression profiles: PRL-3 is apparently expressed in CRC metastases to any organ but is not expressed in metastases of other cancers to the same organs or in nonmetastatic CRCs. PRL-3 is also expressed in tumor vasculature, regardless of the tumor source. These data raise intriguing questions about the role of protein phosphorylation in angiogenesis and cell-type-specific metastatic processes.     

Cancer‐associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor‐β signaling

Cancer‐associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM‐12 and OCUM‐2MD3, and non‐scirrhous gastric cancer cell lines, MKN‐45 and MKN‐74, were used. OCUM‐12/side population (SP) cells and OCUM‐2MD3/SP cells were sorted by flow cytometry as CSC‐rich cells from the parent cells. CaF‐37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT‐PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM‐12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM‐12/SP and OCUM‐2MD3/SP cells, but not that of MKN‐45/SP and MKN‐74/SP cells. Taken together, CM from CaF‐37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM‐12/SP and OCUM‐2MD3/SP cells. These stimulating‐activities by CM were significantly decreased by TGFβ inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM‐12/SP cells with CAFs was significantly high in comparison with that by OCUM‐12/SP cells alone. Phospho‐Smad2 expression level was significantly increased by co‐inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFβ signaling.     

LMO2蛋白在前列腺基质细胞中介导的IL-11、FGF-9旁分泌促进前列腺癌细胞增殖与侵袭

背景与目的：前列腺癌多发生于前列腺外周带,前列腺增生多发于前列腺移行带。前列腺疾病的带性差异机制可能与前列腺组织微环境有关。该研究的前期研究提示,不同区带来源的前列腺基质细胞对上皮细胞的作用存在明显差异,基因芯片筛查发现LMO2蛋白在前列腺外周带基质细胞高表达与前列腺癌发生、发展密切相关。该研究旨在分析前列腺基质细胞LMO2基因的表达对前列腺癌细胞系增殖、侵袭能力的影响及其机制。方法：分别应用慢病毒过表达载体和短发卡RNA(shRNA)建立过表达和低表达LMO2的前列基质细胞,利用实时荧光定量聚合酶链反应(real-time lfuorescent quantitative polymerase chain reaction,RTFQ-PCR)、蛋白[质]印迹法(Western blot)分别检测LMO2 mRNA和蛋白的表达;将不同处理的前列腺基质细胞分别同PC-3细胞共培养,利用CCK-8检测PC-3的增殖能力,利用基质胶侵袭实验检测PC-3的侵袭能力;利用生物素标记的人蛋白抗体芯片检测过表达LMO2的前列腺基质细胞条件培养基中蛋白因子表达变化。结果：成功建立过表达及低表达LMO2的前列腺基质细胞;CCK-8实验及基质胶实验提示,与过表达LMO2的前列腺WPMY-1基质细胞共培养后,PC-3细胞的增殖和侵袭能力增强;与低表达LMO2的CAFs细胞共培养后,PC-3细胞的增殖和侵袭能力降低;蛋白芯片检测发现过表达LMO2后,前列腺外周带基质细胞分泌白介素-11(interleukin-11, IL-11)和成纤维细胞生长因子-9(ifbroblast grouth factor-9,FGF-9)增多。结论：LMO2基因在前列腺外周基质细胞中的高表达可能与前列腺癌的发生、发展有关;过表达LMO2的前列腺基质细胞通过旁分泌IL-11、FGF-9等细胞因子促进前列腺癌细胞增殖与侵袭。     

p16(INK4A) represses the paracrine tumor-promoting effects of breast stromal fibroblasts

Cancer-associated fibroblasts (CAFs), the most abundant and probably the most active cellular component of breast cancer-associated stroma, promote carcinogenesis through paracrine effects; however, the molecular basis remains elusive. We have shown here that p16(INK4A) expression is reduced in 83% CAFs as compared with their normal adjacent counterparts cancer-free tissues isolated from the same patients. This decrease is mainly due to AUF1-dependent higher turnover of the CDKN2A mRNA in CAFs. Importantly, p16(INK4A) downregulation using specific siRNA activated breast fibroblasts and increased the expression/secretion levels of stromal cell-derived factor 1 (SDF-1) and matrix metalloproteinase (MMP)-2. Consequently, media conditioned with these cells stimulated the proliferation of epithelial cells. Furthermore, the migration/invasion of breast cancer cells was also enhanced in an SDF-1-dependent manner. This effect was mediated through inducing an epithelial-mesenchymal transition state. By contrast, increase in p16(INK4A) level through ectopic expression or AUF1 downregulation, reduced the secreted levels of SDF-1 and MMP-2 and suppressed the pro-carcinogenic effects of CAFs. In addition, p16(INK4A)-defective fibroblasts accelerated breast tumor xenograft formation and growth rate in mice. Importantly, tumors formed in the presence of p16(INK4A)-defective fibroblasts exhibited higher levels of active Akt, Cox-2, MMP-2 and MMP-9, showing their greater aggressiveness as compared with xenografts formed in the presence of p16(INK4A)-proficient fibroblasts. These results provide the first indication that p16(INK4A) downregulation in breast stromal fibroblasts is an important step toward their activation.     

Fibroblast growth factor-2, derived from cancer-associated fibroblasts,stimulates growth and progression of human breast cancer cells via FGFR1 signaling

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. In the present study, we investigated the role for CAFs in breast cancer progression and underlying molecular mechanisms. Human breast cancer MDA-MB-231 cells treated with the CAF-conditioned media manifested a more proliferative phenotype, as evidenced by enhanced messenger RNA (mRNA) expression of Cyclin D1, c-Myc, and proliferating cell nuclear antigen. Analysis of data from The Cancer Genome Atlas revealed that fibroblast growth factor-2 (FGF2) expression was well correlated with the presence of CAFs. We noticed that the mRNA level of FGF2 in CAFs was higher than that in normal fibroblasts. FGF2 exerts its biological effects through interaction with FGF receptor 1 (FGFR1). In the breast cancer tissue array, 42% estrogen receptor-negative patients coexpressed FGF2 and FGFR1, whereas only 19% estrogen receptor-positive patients exhibited coexpression. CAF-stimulated MDA-MB-231 cell migration and invasiveness were abolished when FGF2-neutralizing antibody was added to the conditioned media of CAFs. In a xenograft mouse model, coinjection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced tumor growth, and this was abrogated by silencing of FGFR1 in cancer cells. In addition, treatment of MDA-MB-231 cells with FGF2 enhanced expression of Cyclin D1, a key molecule involved in cell cycle progression. FGF2-induced cell migration and upregulation of Cyclin D1 were abolished by siRNA-mediated FGFR1 silencing. Taken together, the above findings suggest that CAFs promote growth, migration and invasion of MDA-MB-231 cells via the paracrine FGF2-FGFR1 loop in the breast tumor microenvironment.     

Expression of galectin‐1 in carcinoma‐associated fibroblasts promotes gastric cancer cell invasion through upregulation of integrin β1

Increased expression of galectin‐1 (Gal‐1) in carcinoma‐associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal‐1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal‐1, and established a co‐culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal‐1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal‐1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal‐1 expression level. A set of cancer invasion‐associated genes were then chosen to identify the possible mechanism of Gal‐1‐induced cell invasion. Among these genes, integrin β1 expression in cancer cells was considered to be associated with Gal‐1 expression. Pre‐blocking of the integrin β1 expression in gastric cancer cells with siRNA could interrupt the invasion‐promoting effect of CAFs with high Gal‐1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal‐1 and integrin β1 expression. Our results showed that high expression of Gal‐1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin β1 expression in gastric cancer.     

SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.     

缝隙连接蛋白26在肺腺癌相关成纤维细胞中的表达及临床意义

目的 探讨缝隙连接蛋白26(connexin 26,Cx26)在肺腺癌相关成纤维细胞中的表达及其临床意义.方法 收集2018年11月至2019年5月于广西医科大学附属肿瘤医院6例手术切除肺腺癌癌组织及相应癌旁组织,分离原代肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)和正常肺...     

Heat shock factor 1 in cancer‐associated fibroblasts is a potential prognostic factor and drives progression of oral squamous cell carcinoma

Heat shock factor 1 (HSF1) is highly expressed in various malignancies and is a potential modulator of tumor progression. Emerging evidence suggests that HSF1 activation in stromal cells is closely related to poor patient prognosis. However, the role of HSF1 in oral squamous cell carcinoma (OSCC) remains elusive. We aimed to investigate the function of HSF1 in cancer‐associated fibroblasts (CAFs) of the tumor microenvironment (TME) and in tumor development. In the present study, we found that HSF1 was highly expressed in both CAFs and tumor cells, and was significantly correlated with poor prognosis and overall survival. Moreover, HSF1 overexpression in CAFs resulted in a fibroblast‐like phenotype of Cal27 cells, induced epithelial‐mesenchymal transition (EMT), and promoted proliferation, migration and invasion in Cal27 cells. HSF1 knockdown attenuated features of CAFs and reduced EMT, proliferation, migration and invasion in Cal27 cells. Furthermore, HSF1 in CAFs promoted tumor growth in nude mice. Taken together, these data suggest that HSF1 expression in CAFs drive OSCC progression, and could serve as an independent prognostic marker of patients with OSCC. Thus, HSF1 is a potent mediator of OSCC malignancy.     

Generation of PRL-3- and PRL-1-specific monoclonal antibodies as potential diagnostic markers for cancer metastases.

PURPOSE: The PRL-3 mRNA is consistently elevated in metastatic samples derived from colorectal cancers. We sought to generate a specific PRL-3 monoclonal antibody (mAb) that might serve as a potential diagnostic marker for colorectal cancer metastasis.EXPERIMENTAL DESIGN: PRL-3 is one of three members (PRL-1, PRL-2, and PRL-3) in a unique protein-tyrosine phosphatase family. Because the three PRLs are 76% to 87% identical in their amino acid sequences, it poses a great challenge to obtain mAbs that are specific for respective phosphatase of regenerating liver (PRL) but not for the other two in the family. We screened over 1,400 hybridoma clones to generate mAbs specific to each PRL member.RESULTS: We obtained two hybridoma clones specifically against PRL-3 and another two clones specifically against PRL-1. These antibodies had been evaluated by several critical tests to show their own specificities and applications. Most importantly, the PRL-3 mAbs were assessed on 282 human colorectal tissue samples (121 normal, 17 adenomas, and 144 adenocarcinomas). PRL-3 protein was detected in 11% of adenocarcinoma samples. The PRL-3- and PRL-1-specific mAbs were further examined on 204 human multiple cancer tissues. The differential expressions of PRL-3 and PRL-1 confirmed the mAbs' specificity.CONCLUSIONS: Using several approaches, we show that PRL-3- or PRL-1-specific mAbs react only to their respective antigen. The expression of PRL-3 in >10% of primary colorectal cancer samples indicates that PRL-3 may prime the metastatic process. These mAbs will be useful as markers in clinical diagnosis for assessing tumor aggressiveness.