Clinical Significance Of Caveolin-1 Expression In Breast Cancer Associated Fibroblasts

Breast cancer is one of the most common tumors of women, accounting for 22% of all female malignant tumors. Compared with the western countries, china is still a low-incidence of breast cancer area, but in recent years has shown rapid increase in morbidity trends, and breast cancer has become the second female malignancy in china. Anti-breast cancer treatments such as surgery, chemotherapy, radiotherapy, endocrine therapy and immunotherapy, great progresses have been made, but the direction of research and treatment is most concentrated on the breast parenchymal cells namely malignant cells. Recently, more and more studies showed that the microenvironment plays a great role in the occurrence of malignant epithelial tumors, which triggers more attention on understanding of stromal cells in prevention of cancer cell aggression and anti-cancer treatment strategy. However, biological characterization of breast cancer stromal cells is not entirely elucidated. Micro-environmental stromal cells surrounding cancer fields are different from mesenchymal cells of normal tissues, which are characterized by:1) extracellular matrix (ECM) components changes; 2) the increase in microvessel density; 3) a large number of inflammatory cell; and 4) fibroblast activation and proliferation. Activated fibroblasts surrounding the cancer lesions, known as the cancer associated fibroblasts (CAFs). Myofibroblasts in desmoplastic reaction named as normal fibroblasts(NFs), they having both fibroblasts and smooth muscle cell properties, are characteristic of myofibroblasts. Myofibroblasts and fibroblasts cannot histological be distinguished. But, the myofibroblasts is ultra structurally characteristic of abundant rough endoplasmic reticulum, muscular filament and dense patch in the cytoplasm, and stress fibers as well as fiber connection on the membrane complex body. Previous studies showed that the CAFs in development of different tumors have the dual roles of defense against cancer cells or promotion for cancer invasion. More and more researches have showed that CAFs activation and proliferation appear to be in order to facilitate the occurrence of tumor cells, proliferation and invasion [11-12]. Therefore, further study of the tumor microenvironment including fibroblasts will help to understand the mechanisms of tumor invasion, metastasis and other biological behavior, and discover new therapeutic targets. Some studies have shown that as target for anti-cancer treatment, CAFs has good value and prospects [8-11]. Main scientific findings are:1.CAFs are the most important components in microenvironment s which can produce ECM (such asⅠ,Ⅱ,Ⅳcollagen and fibronectin) and cathepsin, and a large number of cytokines, adhesion molecules; 2. Normal fibroblasts probably inhibit the malignant transformation of epithelial cells around it, 3. CAFs probably promote tumor formation and affect tumor cell invasion and metastasis model. Usually a-SMA, vimentin, and FAP are considered as markers of myofibroblasts. Because CAFs and NFs in the non-cancer tissue have different environment and probable functions, there are differences in protein expressions between them. Cav-1 is considered to be a tumor suppressor gene [5]. Many studies have shown that Cav-1 protein expression in breast cancer tissue is decreased compared with normal tissue[6-7]. Recent research has further shown that Cav-1 expression in the stroma of breast cancer has more meaningful evaluation on the prognosis than in the epithelium [8-10]. However, Cav-1 expression in stromal CAFs of breast cancer and clinical significance remains unclear. The present study aims mainly on clarifying the different protein expression of CAFs, correlation of Cav-1 expression level with clinic-pathology of invasive breast cancer and establishing new breast cancer risk stratification model combined with stromal CAFs status.Part 1Different Protein Expression Between Cancer Associated fibroblasts and Nonneoplastic myofibroblastsMaterials and Methods73 cases of cancer and non-cancer specimens with desmoplastic reaction retrieved from 2000-2009.The non-cancer specimens were 45.2%(33/73) including nasal polyps, chronic cholecystitis, tubal phlogistic and fiber adenoma, etc. The cancer specimens were 54.8%(40/73) including esophageal, gastric cancer and pancreatic cancer, colorectal cancer, breast cancer etc, were immunohistochemically analyzed. Six proteins generally expressed in stroma:Stromal cell-derived factor-1 (SDF-1), Secreted protein acidic and rich in cysteine(SPARC), Caveolin-1 (Cav-1), Fibroblast growth factor-2 (FGF-2), Tenascin C(TNC) and Osteoblast-specific factor 2(OSF-2). CAFs is defined based on the expression of markers such asα-SMA, and PDGFR-βreceptor, were detected with EnVision method. ResultsCav-1, SPARC and TNC positive rate were 67.1%(49/73)、34.2%(25/73) 47.9%(35/73) respectively. Their difference between CAFs around carcinoma and NFs desmoplastic reaction stroma had a statistically significance. Cav-1 expression pattern in CAFs showed a close approximation to immunoreactivity ofα-SMA positive CAFs.Conclusions1. Increased Cav-1、decreased SPARC and TNC expression in CAFs suggested that there were different proteins between CAFs in cancer stroma and benign lesional myofibroblasts.2. Cav-1 expression pattern in CAFs closely mimicking to the SMA expression suggested Cav-1 should be considered as a ancillary qualifying marker for CAFs.Part 2Clinical Significance of Caveolin-1 Expression in Breast Cancer Associated FibroblastsMaterials and MethodsCav-1 expression in 143 cases of breast cancer was immunohistochemically performed, and its correlation with clinic pathological parameters was analyzed. ResultsCav-1 expression in the stroma of invasive breast cancer showed a close approximation ofα-SMA-positive CAFs, which the difference between two groups was without statistically significance tested by Rank sum test, Cav-1 is a marker of breast cancer associated fibroblasts. The expression rate of stromal Cav-1 in breast cancer 65.7%(94/143) was significantly higher than DCIS 0%(0/10) and normal breast tissue 0%(0/10) (P=0.000); The expression rate of stromal Cav-1 in invasive ductal carcinoma and mixed carcinoma were 74.1%(70/105) and 78.9%(15/19) was significantly higher than that of invasive lobular carcinoma of 47.3%(9/19) (P=0.000, P=0.000). Stromal Cav-1 and ER showed a positive correlation (P=0.046, Spearman correlation coefficient＝0.218); Stromal Cav-1 and histological grade showed a negative correlation (P=0.008, Spearman correlation coefficient=-0.052); Stromal Cav-1 expression level in Molecular typing groups:luminal A was 71.4%(25/35), luminal B was 74.2%(23/31), HER2-type was 66.7%(24/36) and basal cell-like type was 11.1%(1/9), luminal B was significantly higher than basal cell-like type (P=0.048). Log-rank test showed that the age (P=0.000), lymph node metastasis (P=0.042), TNM stage (P=0.027), histological grade (P=0.017) and stromal expression of Cav-1 status (P=0.029) significantly correlated with 5-years survival rate. COX regression analysis showed that Cav-1 expression in stroma was an independent prognostic factor (P=0.009).Conclusion1、Cav-1 expression in stromal levels of CAFs related with breast cancer histological type, histological grade and ER status.2、Cav-1 expression in stromal CAFs of invasive breast cancer was associated with the prognosis, suggesting that reduction of Cav-1 expression in stromal CAFs of invasive breast cancer predict a poor prognostic outcome. Part 3Correlation of Caveolin-1 Stromal Expression with Molecular Subtypes Based on HER2 Gene Amplification of The Invasive Breast CarcinomaMaterials and Methods145 cases of breast cancer molecular subtypes, Luminal A type was 61 cases (42.1%), Luminal B 24 cases (16.6%), HER2+type was 42 cases (28.9%), basal cell-like type 18 cases(12.4%). En Vision immunohistochemical method was used to detect breast cancer in 168 cases of Cav-1 expression in CAFs situation. Fluorescence in situ hybridization (FISH) was used to detect HER2 gene amplification status of breast cancer. Analyze of HER2 gene amplification in breast cancer and stromal expression of Cav-1 correlation.ResultsCav-1 expression in CAFs in breast cancer with pathological closely related to molecular typing, positive rates in HER2+type and Luminal B type groups were 83.3%(35/42)、83.3%(20/24), significantly higher than the Luminal A group57.4%(35/61)and Basal-like type group 38.8%(7/18), the difference Statistically significant (P<0.05). Cav-1 expression in CAFs was positively correlated with HER2 protein expression and HER2 gene status. HER2 gene status were detected by FISH, stromal Cav-1 positive rate of HER2 gene amplification group was 81.8%(54/66) than those without amplification group 55.9%(57/102), the differences were statistically significant(P<0.05). Conclusions1、Cav-1 expression in CAFs in breast cancer closely related to molecular typing, positive rates in HER2 + type and Luminal B type groups are significantly higher than the Luminal A type and Basal-like type groups.2、Cav-1 expression in CAFs is positively correlated with HER2 protein expression and HER2 gene status.