The Mechanism Of Action Of Wnt2/β-catenin Signaling In Breast Carcinoma

Objective1. To investigate the expressions of Wnt2in the cell lines（MDA-MB-231, ZR-75-30and MCF-7）and tissues of breast cancer，andto observe the relation of Wnt2with CA-153in the serum of patients withbreast cancer.2. To investigate the expressions of Wnt2and β-catenin in the matchedpairs of breast cancer and normal breast tissues, and its possible clinicalsignificance.3. To investigate the expression of Wnt2in the carcinoma-associatedfibroblasts (CAFs) and normal fibroblasts (NFs), as well as in their culturesupernatant. At the same time，to observe the effects of different culturesupernatants on the invasion, proliferation, cell cycle of MCF-7, and theexpression and distribution of β-catenin in MCF-7cells.Methods1. The levels of Wnt2mRNA and protein were measured bypolymerase chain reaction (PCR) and Western blotting in3human breastcancer cell lines（MDA-MB-231, ZR-75-30and MCF-7), respectively.2. Enzyme-linked immunosorbent assay (ELISA) was used to examineWnt2, and electrochemiluminescence technique was applied to detect CA-153in the serum of15normal women subjects (control) and30patients with breast cancer. Furthermore, the relation of Wnt2with CA-153was analyzed. The transcription levels of Wnt2and β-catenin mRNA weredetected by RT-PCR in the seven pairs of breast cancer and normal breasttissues. Expressions of Wnt2and β-catenin were also detected with tissuechip technique by immunohistochemistry in the forty pairs of invasivebreast ductal carcinoma and normal breast tissues, and the relation of thetwo protein expressions with clinical and pathological characteristics wasanalized. At the same time, the relationship between parallel combinationdetection of Wnt2and β-catenin with the degree of malignancy of breastcancer and lymph node metastasis was analized.3. Carcinoma-associated fibroblasts (CAFs) and normal fibroblasts(NFs) were isolated from breast cancer tissues and the correspondingadjacent breast tissues，and their culture supernatants were collected,respectively；Wnt2in CAFs and NFs was examined by Western blot, and inthe culture supernatants by ELISA. The following experiments weredivided into4groups: MCF-7control group, NFs culture supernatant+MCF-7group, CAFs culture supernatant containing low Wnt2+MCF-7group, culture supernatant of CAFs containing high Wnt2+MCF-7group.The cell proliferation was detected by the MTT，cell cycle distribution wasassayed by flow cytometry, and cell invasion ability was investigated withMillicell chamber； And expression and intracellular distribution of β-catenin in the MCF-7were detected by immunofluorescence.Results1. The expressions of Wnt2mRNA and protein were negative in threehuman breast cancer cell lines. The levels of Wnt2in the serum of patientswith breast cancer [(10.34±4.25) ng/mL] were higher than those in normalwomen[(1.85±0.98) ng/mL](P<0.05), and Wnt2levels in the serum of breastcancer patients were positively correlated with serum CA-153levels.2. The relative transcription levels of Wnt2and β-catenin gene mRNAwere obviously higher in the breast cancer tissues than adjacent normalbreast tissues（P<0.05)；Wnt2were weakly expressed in the cytoplasm ofepithelial and interstitial cells of some adjacent normal breast tissues，andstrongly expressed in the cytoplasm of epithelial and interstitial cells ofinvasive breast ductal carcinoma. The positive rate of Wnt2expressionswas15.0%(6/40) in the cancer adjacent normal tissues and77.5%(31/40)in the invasive breast ductal carcinoma (P<0.05). β-catenin was onlyexpressed in the cytomembrane of adjacent normal breast tissues，incontrast, it was strongly expressed in the cytomembrane, cytoplasm andnucleus of invasive breast ductal carcinoma. The abnormal expression rateof β-catenin was67.5%（27/40）in invasive breast ductal carcinomain. theexpression levels of Wnt2and β-catenin were correlated with clinical stage,histological granding and lymph nodes metastases (P<0.05)，while nosignificant association was found with patient age (P>0.05). However, the malignant degree and the rate of lymph nodes metastases were higher inpatients with both of Wnt2and β-catenin positive expression than thosewith Wnt2or β-catenin alone expressed.3. CAFs and NFs were separated from four breast cancer tissues andadjacent normal breast tissues, Wnt2was strongly expressed in1of4CAFs (1.33±0.13) and low expressed in other3CAFs (0.22±0.10),however, there was no Wnt2expression in the NFs by Western blotting.Culture supernatant Wnt2test with ELISA showed: Wnt2was high lever[(6.58±1.02) ng/ml] in one culture supernatan of4CAFs, which wasobviously higher than paired NFs [(0.92±0.34)ng/ml] and other3CAFs[(1.67±0.37) ng/ml](all P<0.05); And Wnt2in the culture supernatan ofother3CAFs [(1.67±0.37) ng/ml] was also significantly higher than pairedNFs [(0.54±0.22) ng/ml]（P<0.05).In addition，MTT, FCM and Millicell chamber tests revealed thatcompared with the culture supernatant of NFs, MCF-7hatched with theculture supernatant of CAFs showed significantly higher proliferationability, percentage of S phase cells and invasion ability (all P<0.05);compared with the culture supernatant of CAFs with low Wnt2, MCF-7hatched with the culture supernatant of CAFs with high Wnt2showedsignificantly higher proliferation ability and invasion ability (all P<0.05).Furthermore, compared with the culture supernatant of NFs, β-cateninexpression was increased in the MCF-7hatched with the culture supernatant of CAFs; in addition, compared with the culture supernatant ofCAFs with low Wnt2, the nuclear expression of β-catenin was increased inthe MCF-7hatched with the culture supernatant of CAFs with high Wnt2.Conclusions1. Wnt2cooperating with CA-153can act as markers for breast cancerscreening，parallel combined detection of both may help to improve thesensitivity of breast cancer screening.2. Overexpression of Wnt2and β-catenin in breast carcinoma mayplay some role in the oncogenesis and metastasis of breast cancer. Thecombined detection of Wnt2and β-catenin may be useful makers in thediagnosis and prognosis of breast cancer patients.3. CAFs could promote the proliferation, meiosis and invasion ofbreast cancer cells through Wnt2paracrine, and it may be a novel target ofbreast cancer treatment in the future.