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.     

Cancer‐associated fibroblast promote transmigration through endothelial brain cells in three‐dimensional in vitro models

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood‐brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer‐associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon? Simple Western System to confirm changes in expression levels. We established two novel three‐dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro‐metastasis and colonization. Expression of integrin α5β1 and αvβ3, c‐MET and α2,6‐siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co‐culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis.     

Reverse of non‐small cell lung cancer drug resistance induced by cancer‐associated fibroblasts via a paracrine pathway

The tumor microenvironment orchestrates the sustained growth, metastasis and recurrence of cancer. As an indispensable component of the tumor microenvironment, cancer‐associated fibroblasts (CAF) are considered as an essential synthetic machine producing various tumor components, leading to cancer sustained stemness, drug resistance and tumor recurrence. Here, we developed a sustainable primary culture of lung cancer cells fed with lung cancer‐associated fibroblasts, resulting in enrichment and acquisition of drug resistance in cancer cells. Moreover, IGF2/AKT/Sox2/ABCB1 signaling activation in cancer cells was observed in the presence of CAF, which induces upregulation of P‐glycoprotein expression and the drug resistance of non‐small cell lung cancer cells. Our results demonstrated that CAF cells constitute a mechanism for cancer drug resistance. Thus, traditional chemotherapy combined with insulin‐like growth factor 2 (IGF2) signaling inhibitor may present an innovative therapeutic strategy for non‐small cell lung cancer therapy.     

Podoplanin expression by cancer associated fibroblasts predicts poor prognosis of lung adenocarcinoma

Recent studies have reported increased podoplanin expression by cancer cells and stromal cells, but little is known about its expression and biological significance in adenocarcinoma of the lung. We examined podoplanin expression by both cancer cells and stromal cells in 177 consecutive lung adenocarcinoma cases and analyzed relations between podoplanin expression and both clinicopathological factors and outcome. Podoplanin expression was observed on the apical membrane of the cancer cells in only 9 of the 177 (5.1%) cases. By contrast, cancer-associated fibroblasts (CAFs) were found to express podoplanin in 54 cases (30.5%). Podoplanin (+) CAFs were found only in invasive adenocarcinoma and none were found in noninvasive adenocarcinoma. Conventional prognostic factors were significantly correlated with podoplanin expression by CAFs. The univariate analyses and log-rank test showed that podoplanin expression was significantly associated with shorter survival time (p < 0.001 and p < 0.001, respectively). We divided the cases into 3 groups according grade based on the proportion of CAFs expressing podoplanin [a grade 0 group (n = 123), a grade 1 group (n = 36) and a grade 2 group (n = 18)]. The result showed that conventional prognostic factors were significantly correlated with the grade of podoplanin expression by CAFs. Furthermore, the grade 2 group tended to have a shorter survival time than the grade 1 group (p = 0.092). The results of this study highlight the importance of podoplanin expression by CAFs and provide new insights into the biology of the cancer microenvironment in adenocarcinoma of the lung.     

Expression of long noncoding RNAs in cancer‐associated fibroblasts linked to patient survival in ovarian cancer

Cancer‐associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment and are responsible for producing the desmoplastic reaction that is a poor prognostic factor in ovarian cancer. Long non‐coding RNAs (lncRNAs) have been shown to play important roles in cancer. However, very little is known about the role of lncRNAs in the tumor microenvironment. We aimed to identify lncRNAs expressed in ovarian CAFs that were associated with patient survival and used computational approaches to predict their function. Increased expression of 9 lncRNAs and decreased expression of 1 lncRNA in ovarian CAFs were found to be associated with poorer overall survival. A “guilt‐by‐association” approach was used to predict the function of these lncRNAs. In particular, MIR155HG was predicted to play a role in immune response. Further investigation revealed high MIR155HG expression to be associated with higher infiltrates of immune cell subsets. In conclusion, these data indicate expression on several lncRNAs in CAFs are associated with patient survival and are likely to play an important role in regulating CAF function.     

Upregulated microRNA‐193a‐3p is responsible for cisplatin resistance in CD44(+) gastric cancer cells

Cisplatin is a well‐known anticancer drug used to treat various cancers. However, development of cisplatin resistance has hindered the efficiency of this drug in cancer treatment. Development of chemoresistance is known to involve many signaling pathways. Recent attention has focused on microRNAs (miRNAs) as potentially important upstream regulators in the development of chemoresistance. CD44 is one of the gastric cancer stem cell markers and plays a role in regulating self‐renewal, tumor initiation, metastasis and chemoresistance. The purpose of the present study was to examine the mechanism of miRNA‐mediated chemoresistance to cisplatin in CD44‐positive gastric cancer stem cells. We sorted gastric cancer cells according to level of CD44 expression by FACS and analyzed their miRNA expression profiles by microarray analysis. We found that miR‐193a‐3p was significantly upregulated in CD44(+) cells compared with CD44(?) cells. Moreover, SRSF2 of miR‐193a‐3p target gene was downregulated in CD44(+) cells. We studied the modulation of Bcl‐X and caspase 9 mRNA splicing by SRSF2 and found that more pro‐apoptotic variants of these genes were generated. We also found that downstream anti‐apoptotic genes such as Bcl‐2 were upregulated, whereas pro‐apoptotic genes such as Bax and cytochrome C were downregulated in CD44(+) cells compared to CD44(?) cells. In addition, we found that an elevated level of miR‐193a‐3p triggered the development of cisplatin resistance in CD44(+) cells. Inhibition of miR‐193a‐3p in CD44(+) cells increased SRSF2 expression and also altered the levels of multiple apoptotic genes. Furthermore, inhibition of miR‐193a‐3p reduced cell viability and increased the number of apoptotic cells. Therefore, miR‐193a‐3p may be implicated in the development of cisplatin resistance through regulation of the mitochondrial apoptosis pathway. miR‐193a‐3p could be a promising target for cancer therapy in cisplatin‐resistant gastric cancer.     

Microtubule affinity‐regulating kinase 2 is associated with DNA damage response and cisplatin resistance in non‐small cell lung cancer

Microtubule affinity‐regulating kinases (MARKs) are involved in several cellular functions but few studies have correlated MARK kinase expression with cancer, and none have explored their role in lung cancer. In this study, we identified MARK2 as frequently disrupted by DNA hypomethylation and copy gain, resulting in concordant overexpression in independent lung tumor cohorts and we demonstrate a role for MARK2 in lung tumor biology. Manipulation of MARK2 in lung cell lines revealed its involvement in cell viability and anchorage‐independent growth. Analyses of both manipulated cell lines and clinical tumor specimens identified a potential role for MARK2 in cell cycle activation and DNA repair. Associations between MARK2 and the E2F, Myc/Max and NF‐κB pathways were identified by luciferase assays and in‐depth assessment of the NF‐κB pathway suggests a negative association between MARK2 expression and NF‐κB due to activation of non‐canonical NF‐κB signaling. Finally, we show that high MARK2 expression levels correlate with resistance to cisplatin, a standard first line chemotherapy for lung cancer. Collectively, our work supports a role for MARK2 in promoting malignant phenotypes of lung cancer and potentially modulating response to the DNA damaging chemotherapeutic, cisplatin.     

NPPB is a novel candidate biomarker expressed by cancer‐associated fibroblasts in epithelial ovarian cancer

Most solid tumors contain cancer‐associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF‐like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF‐specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.     

Akt1 inhibition by RNA interference sensitizes human non-small cell lung cancer cells to cisplatin

Akt/protein kinase B signaling is very important for cancer cell survival and growth when cells are exposed to various apoptotic stimuli. Akt is constitutively activated in NSCLC cells and is a potential target for enhancing the cytotoxicity of chemotherapeutic agents in treatment of NSCLC. In our study, we investigated whether down-regulating Akt1 using RNAi techniques can enhance sensitivity to cisplatin in NSCLC cells. An siRNA targeting Akt1 significantly decreased the protein level of Akt1 and the activity of ERK. Treatment of these cells with 20 muM cisplatin increased apoptotic cell death approximately 2.6-fold compared to cells transfected with a scrambled siRNA. While Akt activity was slightly reduced, ERK activity was greatly increased in cells treated with cisplatin alone. Pretreatment of these cells with the selective MEK inhibitor U0126 effectively reduced the level of cisplatin-induced apoptosis. These results imply that cisplatin-induced MEK/ERK activation appears to mediate apoptotic cell death, but that constitutively activated Akt1 and/or ERK pathway may mediate resistance to cisplatin in NSCLC cells. Taken together, our data demonstrate that down-regulation of Akt1 using RNAi enhances the chemosensitivity of NSCLC cells to cisplatin.     

Downregulation of miR‐29 contributes to cisplatin resistance of ovarian cancer cells

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin‐resistant cells expressed a lower level of miR‐29a/b/c. Manipulation of microRNA‐29 (miR‐29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR‐29a/b/c increased the ability of cells to escape cisplatin‐induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal‐regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR‐29 decreased the amount of the active form of caspase‐9 and caspase‐3. Ectopic expression of miR‐29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR‐29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR‐29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.     

Cancer‐associated fibroblasts and epithelial‐mesenchymal transition in metastatic oral tongue squamous cell carcinoma

We examined cancer‐associated fibroblasts (CAFs) and a panel of immunohistochemical markers of epithelial‐mesenchymal transition (EMT) in 19 pair‐matched oral tongue squamous cell carcinoma (SCC) and metastatic tumors to regional lymph nodes (RLNs). α‐Smooth muscle actin (α‐SMA) was studied to identify CAFs. EMT was studied with syndecan‐1, Cadherin‐11, fibroblast‐specific protein (FSP)‐1, s ecreted p rotein a cidic and r ich in c ysteine (SPARC) and Twist. Triple immunostaining in RLNs was used to highlight the carcinoma cells (E‐cadherin and Ki‐67) and their relationship to the CAFs (α‐SMA). We found that metastatic RLNs hosted CAFs similarly as in pair‐matched primary tumors. Expression of EMT markers is common in both primary and metastatic tumors. We demonstrate that metastatic carcinoma cells (Ki‐67 positive) downregulate E‐cadherin expression at the periphery of cancer islands, where they are in direct contact with CAFs. The supporting connective tissue microenvironment also commonly expresses syndecan‐1, Cadherin‐11, FSP‐1, and SPARC. In conclusion, CAFs are common to both primary and metastatic SCC. We hypothesize that CAFs not only promote tumor invasion but also facilitate metastases, either by cometastasizing and/or being recruited to lymph nodes. Evidence of EMT is common within primary tumors and metastatic SCC and may be further modulated by CAFs.     

Prognostic significance of carbonic anhydrase IX expression by cancer‐associated fibroblasts in lung adenocarcinoma

BACKGROUND:

Cancer tissue is comprised of cancer cells and several types of stromal cells, including cancer‐associated fibroblasts (CAFs). Carbonic anhydrase (CA) IX has been used as an endogenous hypoxia marker, and although its expression by cancer cells has been reported to be associated with a poor outcome in a board range of tumors, to the authors' knowledge, the biologic significance of its expression by CAFs remains unclear.

METHODS:

The authors investigated CA IX expression by CAFs and cancer cells immunohistochemically in 158 consecutive resected cases of lung adenocarcinoma.

RESULTS:

CA IX was expressed by CAFs in 39 (24.7%) of the 158 cases and by cancer cells in 40 (25.3%) cases. CA IX expression by CAFs was found to be significantly correlated with conventional prognostic factors, including pathologic tumor classification and lymph node involvement. A univariate analysis and the log‐rank test demonstrated a significant association between CA IX expression by CAFs (P = .006 and P = .0052, respectively) and by cancer cells (P = .020 and P = .0179, respectively) with lower survival rate. A multivariate analysis of these 2 factors indicated a statistically significant association between CA IX expression by CAFs and a lower survival rate (hazards ratio [HR], 1.797; P = .032), but not between expression by cancer cells and lower survival rate (HR, 1.561; P = .102).

CONCLUSIONS:

The findings of the current study indicate that CA IX expression by CAFs was a better predictor of outcome than CA IX expression by cancer cells and provides new insights into the biologic significance of CAFs in the hypoxic microenvironment of the lung adenocarcinoma. Cancer 2009. © 2009 American Cancer Society.     

Cancer‐associated adipocytes promote pancreatic cancer progression through SAA1 expression

Although pancreatic cancer often invades peripancreatic adipose tissue, little information is known about cancer‐adipocyte interaction. We first investigated the ability of adipocytes to de‐differentiate to cancer‐associated adipocytes (CAAs) by co‐culturing with pancreatic cancer cells. We then examined the effects of CAA‐conditioned medium (CAA‐CM) on the malignant characteristics of cancer cells, the mechanism underlying those effects, and their clinical relevance in pancreatic cancer. When 3T3‐L1 adipocytes were co‐cultured with pancreatic cancer cells (PANC‐1) using the Transwell system, adipocytes lost their lipid droplets and changed morphologically to fibroblast‐like cells (CAA). Adipocyte‐specific marker mRNA levels significantly decreased but those of fibroblast‐specific markers appeared, characteristic findings of CAA, as revealed by real‐time PCR. When PANC‐1 cells were cultured with CAA‐CM, significantly higher migration/invasion capability, chemoresistance, and epithelial‐mesenchymal transition (EMT) properties were observed compared with control cells. To investigate the mechanism underlying these effects, we performed microarray analysis of PANC‐1 cells cultured with CAA‐CM and found a 78.5‐fold higher expression of SAA1 compared with control cells. When the SAA1 gene in PANC‐1 cells was knocked down with SAA1 siRNA, migration/invasion capability, chemoresistance, and EMT properties were significantly attenuated compared with control cells. Immunohistochemical analysis on human pancreatic cancer tissues revealed positive SAA1 expression in 46/61 (75.4%). Overall survival in the SAA1‐positive group was significantly shorter than in the SAA1‐negative group (P = .013). In conclusion, we demonstrated that pancreatic cancer cells induced de‐differentiation in adipocytes toward CAA, and that CAA promoted malignant characteristics of pancreatic cancer via SAA1 expression, suggesting that SAA1 is a novel therapeutic target in pancreatic cancer.     

Pyruvate secreted from patient‐derived cancer‐associated fibroblasts supports survival of primary lymphoma cells

Cancer‐associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor‐cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse‐Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells.