| Abstract |
Angiogenesis is a complex and multistep process leading to the formation of new blood vessels from the pre-existing vascular network. Recent experimental and clinical data have demonstrated that angiogenesis is essential for tumor development and metastasis. Tumor growth more than 1-2mm is solely dependent on angiogenesis. The mechanism that controls the angiogenic potential of a tumor remains unclear. It is believed that either up regulation of angiogenic growth factors, or down regulation of naturally occurring inhibitors, leads to the disruption of their net balance and initiates the angiogenic switch. Environmental and genetic changes seem to affect the angiogenic process as well. The role of angiogenesis has been described in all human cancers including breast and cervical cancer. It is believed to play an accompanying role in carcinogenesis and it is suggested to comprise a marker of malignant transformation. The purpose of the present study is to investigate the mechanism that controls the induction of the angiogenic process in the most frequent female cancers, as well as to determine similarities or differences in the procedure that accompanies the malignant conversion of normal breast and cervical epithelial cells. Therefore, we evaluated the mRNA expression of VEGF, bFGF, TGF-β1, β2, β3 and the receptors TGF-β RI, RII, RIII by RT-PCR in breast cancer tissue samples and adjacent normal specimens. We also evaluated the protein levels of VEGF and TGF-β1 (western blot analysis), and we examined the correlation with their mRNA expression levels. Furthermore, the mRNA expression profile of the same growth factors was investigated in tissue samples with cervical intraepithelial neoplasia (CIN) and cervical cancer and compared to that of normal cervical tissues. Our goals were: a) to determine the mRNA expression profile of the main growth factors implicated in angiogenesis, b) to examine the possible correlation of the mRNA expression profile with the clinical stage of the disease, or other clinicopathological features of the specimens, and c) to examine the possibility of using the mRNA expression profile of growth factors as a molecular marker of angiogenesis and malignant transformation of epithelial cells. Our results show significant differences in TGF-β1 and TGF-β3 mRNA levels in breast cancer specimens of differing histology (ductal, lobular, other) (P=0.020 and P=0.043). VEGF mRNA was elevated in tumor specimens compared to controls but the increase was not statistically significant. VEGF protein in tumors was associated with patients menopausal status. A strong hormonal influence of ER and PR on TGF-β mRNA expression was established. FGF2 transcript levels were substantially decreased in cancer compared to adjacent normal specimens (P=0.031). Western blot analysis revealed differences between VEGF and TGFβ1 mRNA and protein levels. A substantial negative correlation of TGF-β1 protein and TGF-β1 mRNA levels (p=0.016) was demonstrated by breast tissue-pair analysis. A disruption of mRNA co-expression patterns was observed in malignant breast tissues compared to controls. VEGF, TGFB1, TGFBR1, and FGF2 transcript levels were significantly different in the normal, CIN and cancer specimen groups (P=0.015, 0.001, 0.008, and 0.029 respectively). Higher TGFBR1 mRNA levels were observed in parallel with increased severity of the lesion, whereas FGF2 exhibited lower transcript levels. A highly significant increase of VEGF mRNA expression was found upon cervical neoplastic transformation (P<0.0001). High-grade squamous intraepithelial lesions exhibited higher VEGF mRNA levels than low-grade lesions (P=0.039). TGFBR1 and TGFBR3 receptors demonstrated significant co-expressions with TGFB2 (P<0.0001), and TGFB1 (P=0.005 and P=0.002 respectively) in normal cervical specimens. However, a disruption of co-expression patterns was observed in the groups of CIN and cancer cases, compared to normal tissues. Our results in breast and cervical cancer indicate that different growth factors prevail in the angiogenic process accompanying the malignant transformation of epithelial cells in each type of cancer. The disruption of VEGF, bFGF, TGF-β1, and TGF-β RI mRNA expression is implicated in cervical carcinogenesis, whereas only bFGF and TGF- β3 mRNA expression was significantly different in the groups of cancer and adjacent normal breast tissues. Correlation of mRNA expression levels of the growth factors studied with clinicopathological features of the specimens, revealed differences in breast and cervical cancer suggesting that angiogenesis is context dependent. A common feature of both types of cancer however, is the disruption of mRNA co-expression patterns of all growth factors included in our evaluation, in cancer (and CIN lesions) compared to normal specimens. The latter provides additional evidence that tumor development is accompanied by the disruption of the net balance of angiogenic growth factors and naturally occurring inhibitors. Our findings suggest that the mRNA expression profile of growth factors could possibly be used as a molecular marker of angiogenesis and a marker of malignant conversion of normal epithelial cells in female cancers.
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Μελέτη του φαινομένου της αγγειογένεσης στην εμφάνιση και εξέλιξη του καρκίνου του μαστού και του τραχήλου της μήτρας
