Abstract
Background: Particularly interesting new cysteine-histidine-rich protein (PINCH) was found to be up-regulated in the stroma of colorectal carcinomas (CRCs) in our previous studies and was involved in angiogenesis through activation of fibroblasts in extracellular matrix (ECM) in response to tumors. Here, we examined PINCH mRNA expression in colorectal cancer and investigated its relationship with the clinicopathological features and proliferation cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF) and FAS. Materials and Methods: The primary cancer tissues, adjacent noncancerous tissues and the proximal and distant margins of normal mucosa were collected from 81 colorectal cancer patients during surgery. PINCH, PCNA, VEGF and FAS mRNA expression was examined by reverse transcriptional PCR (RT-PCR). Results: PINCH mRNA expression was significantly increased in primary tumors compared with that in adjacent noncancerous tissues, and the proximal and distant margins of normal mucosa (p<0.0001). Expression of PINCH mRNA in colon cancer tended to be higher than expression in rectal cancer (p=0.051). Tumors which had infiltrated through the wall of the colorectum trended to have higher PINCH mRNA expression (p=0.073). PINCH mRNA expression in primary tumors was positively related to the expression of PCNA mRNA (r=0.534, p=0.010), VEGF mRNA (r=0.431, p=0.022) and FAS mRNA (r=0.542, p=0.012). Conclusion: PINCH mRNA was overexpressed in colorectal cancer and associated with PCNA mRNA, VEGF mRNA and FAS mRNA expression. PINCH mRNA was involved in the development of colorectal cancer and might play a role in the epithelial mesenchymal transition in the rectum differently than in the colon, through the adenomatous polyposis coli (APC)/catenin pathway.
- Cysteine-histidine-rich protein (PINCH)
- colorectal carcinoma
- epithelial mesenchymal transition (EMT)
- vascular endothelial growth factor (VEGF)
- FAS
Colorectal cancer (CRC) is the third most common cancer worldwide, with an estimated one million new cases and a half million deaths each year (1). Although the 5-year mortality rate of colorectal cancer has declined over the last three decades, it is still necessary to identify more effective prognostic biomarkers and therapeutic targets. Particularly interesting new cysteine-histidine-rich protein (PINCH) is a widely expressed, evolutionarily conserved protein that consists of five Lin-1, Isl-1, Mec-3 (LIM) domains and an auto-epitope homologous to ‘senescent cell antigen’ (2). PINCH is located on chromosome 2q12.2, and the protein functions as an adapter protein for signal transduction in the integrin and growth factor pathways (3-5). PINCH mRNA is expressed in most normal tissues (2). PINCH protein has been shown to be markedly overexpressed in the tumor-associated stroma in a variety of common human cancer types compared with the corresponding normal tissues, including breast, prostate, lung, skin, and colon cancer (6). In our previous studies, the expression of PINCH protein was not only up-regulated in the stroma of the primary tumors and lymph node metastases of colorectal cancer, but also in mucinous and signet-ring cell carcinomas compared to non-mucinous carcinomas (7). However, there was no information available on PINCH mRNA expression in the primary tumor of colorectal cancer.
To understand the role of PINCH mRNA in colorectal adenocarcinoma, we examined the expression of PINCH mRNA in primary tumors, the non-cancerous mucosa samples which were 2 cm away from tumors, and the distant normal mucosal samples of proximal and distal margins, and analyzed the relationship between PINCH mRNA expression and the clinicopathological variables of colorectal cancer, including patient's gender, age, tumor location, histological type, grade of differentiation, invasive depth, lymph node metastasis and Dukes' stage. Furthermore, we analyzed the relationship between PINCH mRNA expression and proliferation cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF) and FAS mRNA expression.
Materials and Methods
Patients. Fresh cancer samples were obtained from 81 patients with colorectal cancer during surgeries between 2007 and 2008 and immediately placed in liquid nitrogen at Tangshan Gongren Hospital. The adjacent non-cancerous mucosa specimens 2 cm away from tumors, and normal mucosal specimens of proximal and distal margins were collected at the same time. All the patients gave signed informed consent. None of the patients had received preoperative radiotherapy or chemotherapy. The patient's gender, age, tumor location, histological type, grade of differentiation, invasive depth, and lymph node metastasis were obtained from surgical and pathological records. Patients with incomplete clinical records were excluded from certain analyses. The mean age of the patients was 57.6 years (range from 31-90 years). The histology of the tumors was as follows: non-mucinous adenocarcinoma (n=51), mucinous/ signet-ring cell carcinomas (n=24). Tumors were graded as well (n=5), moderately (n=53) and poorly (n=20) differentiated. All slides of normal mucosa specimens and tumors were examined by two pathologists (Z.Y. Zhang and Y. M. Hu). In addition, cases including primary tumors, adjacent noncancerous mucosa specimens and normal mucosal specimens of proximal and distal margins were chosen randomly for the detection for PCNA mRNA (n=24), FAS mRNA (n=24) and VEGF mRNA (n=29).
Total RNA extraction. Total RNA was extracted from tissue samples using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Fresh tissue was homogenized in 1 ml TRIzol to isolate total RNA.
Reverse transcription-polymerase chain reaction. Total RNA (2 μg) was reverse transcribed into cDNA using the M-MLV Reverse Transcription System (Promega, San Luis Obispo, CA, USA). A DNA-free template control (containing water) was included and each sample was reverse transcribed in duplicate. To quantify the expression of the target gene, 10 μl samples of PCR products were separated by 1.5% agarose gel electrophoresis and quantified by Gel Imaging System (TFP-M/WL, Vilber Lourmat, France) after ethidium bromide (10 mg/l) staining. The mRNA expression was estimated by the densitometric ratio of the target gene to β-actin. The primers for PINCH, PCNA, FAS and VEGF, and the conditions of RT-PCR are shown in Table I.
Statistical analysis. All data are presented as mean±SD and analyzed by SPSS 15.0 (SPSS Inc., Chicago, IL, USA). Unpaired and paired Student's t-test and one-way ANOVA were used to test the relationship between PINCH mRNA expression and other clinicopathological variables. Pearson's correlation test analyzed the relationship between PINCH mRNA expression and PCNA, FAS and VEGF mRNA expressions. A p-value <0.05 was considered as statistically significant.
Primers and thermal cycle conditions used (n=35 cycles).
Results
The expression of PINCH mRNA in primary tumor, adjacent non-cancerous mucosa, and distant normal mucosa of proximal and distal margins. The expression of PINCH mRNA in primary tumors was notably higher than its expression in adjacent non-cancerous mucosa, and distant normal mucosa of proximal and distal margins (all p<0.0001, Figure 1A and B). Similar results were obtained by electrophoresis and densitometric analysis. As shown in Figure 1A, PINCH signal in the primary tumor lane was significantly stronger than in lanes loaded with PCR products from the adjacent noncancerous tissue or the proximal and distant margin of normal mucosa. There was no significant difference of PINCH mRNA expression between adjacent non-cancerous mucosa and distant normal mucosa of proximal or distal margins (p>0.05, Figure 1B).
The relationship between PINCH mRNA expression in primary tumor and other clinicopathological variables. As shown in Table II, the expression of PINCH mRNA in colon carcinomas tended to be higher than that in rectal carcinomas (0.816±0.51 vs. 0.635±0.293, p=0.051). Tumors infiltrating through the wall of the colorectum tended to have higher PINCH mRNA expression (0.554±0.324 vs. 0.747±0.417, p=0.073). There was no correlation between PINCH mRNA expression and other clinicopathological variables including gender (p=0.319), age (p=0.823), histological type (p=0.727), tumor differentiation (p=0. 924), lymph node metastasis (p=0.569) and Dukes' stage (p=0.171).
RT-PCR of PINCH mRNA expression in primary tumor, adjacent noncancerous mucosa, and proximal and distant margins of normal mucosa. A: Lane M, 1, 2, 3 and 4 were marker, tumor, adjacent noncancerous mucosa, proximal, and distant margins of normal mucosa, respectively. β-Actin (378 bp) was used as control. B: Quantfication of PINCH (199 bp) (A) mRNA expression from cancer was stronger than that from adjacent noncancerous mucosa, and that from the proximal and distant margins of normal mucosa.
The relationship between the mRNA expressions of PINCH, and PCNA, VEGF and FAS in colorectal cancer. The mRNA expressions of PCNA, VEGF and FAS in primary tumors was higher than that in the adjacent non-cancerous mucosa, and distant normal mucosa of proximal and distal margins (p<0.0001) (Figure 2). PINCH mRNA expression was positively related to the expression of PCNA mRNA (r=0.534, p=0.01), VEGF mRNA (r=0.431, p=0.022) and FAS mRNA (r=0.542, p=0.012, Figure 3). There was no significant difference of PCNA, VEGF and FAS mRNA expressions between adjacent non-cancerous mucosa and distant normal mucosa of proximal and distal margins (p>0.05, data not shown).
Discussion
It has been widely accepted that the initiation and progression of tumors are influenced by the behavior of tumor microenvironments comprising the extracellular matrix (ECM), the newly formed vasculature, inflammatory cells and fibroblasts (8, 9). Tumor-associated fibroblasts (activated fibroblasts and myofibroblasts) have a well-recognized role as a source of paracrine (cell to cell) growth factors that influence the growth, migration and invasion of carcinoma cells in the carcinogenic process. Activated fibroblasts are responsible for the synthesis, deposition and remodeling of ECM in tumor stroma (8). PINCH is expressed in activated fibroblasts and myofibroblasts and has been implicated as a ‘molecular switch’ for multiple protein-protein interactions mediating integrin signals within focal adhesions (3, 10). PINCH protein expression was found to be up-regulated in the tumor-associated stroma in a variety of human cancer types compared with the corresponding normal tissues (6). PINCH protein expression was higher in oral squamous cell carcinomas, esophageal squamous cell carcinomas, gliomas and colorectal cancer than that in the normal tissues in our previous studies (7, 11-14). In the present study, the PINCH mRNA was also overexpressed in primary tumor compared with that in the adjacent non-cancerous mucosa, and distant normal mucosa of proximal and distal margins. It seems that PINCH mRNA is also involved in the tumorigenesis of colorectal cancer.
PINCH mRNA expression in relation to clinicopathological variables of colorectal cancer.
In the present study, PINCH mRNA expression tended to be higher in cases where the tumor cells infiltrated throughout the wall. The infiltrative growth pattern is associated with recurrence and unfavorable prognosis in colorectal cancer. This demonstrates that PINCH mRNA appears to be related to aggressive features during the development of colorectal cancer. The expression of PINCH mRNA was somewhat related to the location of primary tumor in the present study: tumors in the colon trended to have higher PINCH mRNA expression. Different genetic abnormalities have been found in colorectal carcinomas from different sites. Colon cancer and rectal cancer are different entities with molecular and biological differences. The relationship between the expression of PINCH mRNA and tumor location lends further evidence to this. A family history of colorectal cancer appeared to affect relative risk of colon cancer more strongly than relative risk of rectal cancer (15). Rectal carcinomas exhibited significantly more nuclear β-catenin than colon carcinomas, which is a critical mediator of the WNT signaling pathway negatively regulated by adenomatous polyposis coli (APC) (16). Additionally, compared with colon cancer, rectal cancer is less KRAS-dependent and the APC gene-restricting pattern is more common (17). Therefore, the APC/catenin pathway in rectal cancer was suggested as the predominant one. Huang et al. found SW480 colon cancer cells to overexpress the gene encoding thymosin β4 (Tβ4), which was accompanied by a loss of E-cadherin, as well as a cytosolic accumulation of β-catenin, two most prominent markers of epithelial mesenchymal transition (EMT). Integrin-linked kinase (ILK) up-regulation in Tβ4- overexpressing SW480 cells seemed to be attributed mainly to a stabilization of this kinase by complexing with PINCH more efficiently (18). In this context, we suggest that the expression of PINCH mRNA in the rectum might indirectly be regulated by carcinogenetic pathway, e.g. by APC/WNT pathway, differently from that in the colon. However, more experimental evidence is required for this.
PINCH belongs to a family of cell ECM adhesion proteins involved in regulating cellular proliferation, differentiation and survival by interaction with ILK, participating in integrin-mediated intracellular and growth factor signaling pathways. Moreover, recent studies have raised the concept of the co-evolution of tumor cells with tumor-associated stroma. The stromal environment of tumors appears to be a leading factor, and not just a supporting one in the initiation of tumors (19). The tumor microenvironment and interactions between tumor and stromal cells have a reciprocal relationship in tumor development and progression. EMT is essential for normal embryonic development, but also for progression of non-invasive adenomas into malignant, metastatic carcinomas. Alterations in cell-cell adhesion, cell-substrate interaction, ECM degradation and cytoskeleton organization are major events that occur during EMT (20). In one of our previous studies, endosialin overexpressed on fibroblasts in the tumor-associated stroma in colorectal cancers as well as PINCH protein overexpressed on the fibroblasts in the stroma. But we found a few cases with endosialin expressed on tumor cells. This hinted the interactions of endosialin between the tumor microenvironment and tumors (21). Thus, we further hypothesize that PINCH might play a role in the EMT in the rectum different from that in the colon through the WNT and APC pathway. If therapies target on PINCH, they might block the signal transduction both in stroma and tumor cells, providing more efficient strategies for therapy of patients with rectal cancer.
RT-PCR of PCNA, VEGF and FAS mRNA expression in primary tumor, adjacent noncancerous mucosa, and proximal and distant margins of normal mucosa. A: Lane M, 1, 2, 3 and 4 were marker, tumor, adjacent noncancerous mucosa, and proximal and distant margins of normal mucosa, respectively. β-Actin (378 bp) was used as control. B: Quantfication of PCNA (206 bp) (A), VEGF (246 bp) and FAS (189 bp) mRNA expression from cancer was stronger than that of adjacent noncancerous mucosa, and that of the proximal and distant margins of normal mucosa.
It has been determined that poor differentiation, abundant necrosis, and increased PCNA and VEGF expression represent a more malignant potential of tumors (22). There was also a positive relationship found between PCNA and apoptosis (23). FAS is an important receptor to induce apoptosis, activated by FAS ligand in the target cell surface. FAS/FASL are closely related to the infiltration and metastasis of tumor. VEGF is highly expressed in several types of human solid tumors including colorectal, breast, lung and prostate carcinoma through improved tumor neovascularity and the proliferation of tumor cells (24). It has been shown in vitro and in vivo that ILK is implicated in the promotion of tumor angiogenesis by stimulating VEGF expression (25).
PINCH expression was particularly intense in stromal cells at invasive edges that can facilitate cancer invasion (6, 13). In previous study, endothelial cells that stained for CD31 co-stained for PINCH, indicating that some endothelial cells in the tumor vasculature expresses PINCH protein (13). We showed that strong expression of PINCH was associated with high microvessel density in stroma of colorectal tumors (26). All these data demonstrate that PINCH seems to be involved in angiogenesis through activation of fibroblasts in the ECM in response to tumors. In the present study, PINCH mRNA overexpression in primary tumors was positively related to PCNA, VEGF and FAS mRNA expression, indicating PINCH might participate in tumor development associated with the regulation of proliferation, angiogenesis and apoptosis.
The relationship between mRNA expressions of PINCH and PCNA, VEGF and FAS. PINCH mRNA expression was positively related to PCNA (A), VEGF (B) and FAS(C) mRNA expressions.
Conclusion
Our findings suggest PINCH mRNA expression may play a role in the EMT in the rectum different from in the colon through the APC/catenin pathway.
Acknowledgements
The Authors would like to thank senior nurse Yan M. Gao for collecting samples and Doctor Hai X. Jiang for making the database. The study was supported by a grant from the Chinese National Natural Science Foundation (No. 30772115).
Footnotes
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Competing Interests
The Authors declare that they have no competing interests.
- Received September 21, 2011.
- Revision received November 14, 2011.
- Accepted November 15, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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