Target HER2 And UPAR Genes To Therapy Breast Cancer

Breast cancer is one of the most frequent malignant tumors in female. There are about 120 million women suffering from breast cancer, about 50 million women die of this disease all of the world each year. In recent years, the morbidity of breast cancer was increased sharply in the cities of China. Breast cancer is a systemic disease, the recurrence and metastasis rates are very high after surgical operation, and even the primary tumors have the possibility of metastasis. There are no effective therapy methods for breast cancer after the tumor metastasis to a distant organ. Therefore, to study the pathogenesis of cancer, explore the diagnostic and prognostic biomarkers, and find new therapeutic targets is an important oncology research content in today.HER2 is an important oncogene, its expression product belongs to the epidermal growth factor receptor (EGFR) family. The EGFR family includes four members of HER1, HER2, HER3 and HER4. All of the members have similar moecule structures and functions. EGFR family members binding with ligands to form homo-dimers or hetero-dimers activate intracellular tyrosine kinase, which further activate cysto-signal transduction pathway. EGFR family mediate four mainly cysto-signal transduction pathways, which regulate diverse biological responses, such as proliferation, differentiation, cell motility, and survival.Urokinase-type plasminogen activator receptor (uPAR) is highly glycosylated, extracellular protein, be attached to the cell surface by a GPI anchor. CD87 is a broadly expressed protein, including expression in tumor tissue and cell lines. In tumor cells, uPAR over-expression indicates the worsening of malignant tumors and poor prognosis. uPAR is a multifunctional surface molecular and has the principle function of retaining and concentrating uPA at the cell surface for conversion of plasminogen to plasmin, which exerts its primary role of pericellular proteolysis by activating several metalloproteinases. This process is critical for chemotaxis and cell migration. uPAR can interact with some cell surface factors (integrin formyl Methionine receptor and Vitronectin) and play an important role in cell adhesion, proliferation, apoptosis, and tumor dormancy.Herceptin is a recombinant humanized monoclonal antibody against the HER2 protein that can specifically recognize and bind to the extracellular domain of HER2 molecule. The mechanisms of the action of this antibody involve inhibition of signal transduction. As the first molecular drug, which was approved by FDA, Herceptin bring a new break-through for breast cancer clinical treatment, especially for HER2/neu positive breast cancer patients. Clinical study confirmed that Herceptin mainly was used to treat HER2 over-expression breast cancer patients. When Herceptin was used to cure HER2 gene amplification patients alone, the efficiency is about 25%; when Herceptin combined with chemotherapy, the efficiency is about 50%. After treatment of Herceptin, the average survival period of patients can be extended one year. However, for HER2 low expression breast cancer group, Herceptin has a poor treatment outcome.Previous studies have shown HER2 and uPAR expression exist mutually influence. So, we used RNAi technology to study if HER2 and uPAR could regulate protein expresson mutually; and explore synergistic therapy for breast cancer targeting to HER2 and uPAR.(1) With human HER2 and uPAR gene as a target gene, design and construct the specificity RNA interference plasmid vectors pHER2, puPAR and pHu. pHER2 and puPAR target to HER2 and uPAR respectively; Among them, pHu target both HER2 and uPAR. Then, use real time PCR and western blotting to detect the effect of inhibition target genes expression. The results displayed that we successfully constructed four RNAi plasmids. pHER2 and puPAR can inhibite HER2 or uPAR gene expression respectively in breast cancer cells; and pHu can inhibit both HER2 and uPAR genes expression. Compare pHu with pHER2 or puPAR, pHu has a high efficiency of inhibition HER2 or uPAR gene expression in breast cancer cells.(2) Transfect pHER2, puPAR and pHu to breast cancer cells, and use real time PCR, western blotting, and IF to detect HER2 and uPAR genes expression levels in each transfection group. The results showed that transfected pHER2 to breast cancer cells not only strongly inhibited HER2 expression, but also partly inhibited uPAR expression. Likewise, when puPAR was transfected to breast cancer cells, not only strongly inhibited uPAR expression, but also partly inhibited HER2 expression. Compare pHu with pHER2 or puPAR, pHu has prominence efficiency in inhibition both HER2 and uPAR genes expression.(3) pHER2, puPAR and pHu was transfected to breast cancer cells, then detected and analyzed breast cancer cells proliferation, apoptosis, cycles, invasiveness, and regulation of MAPK signaling pathway activation. The results displayed that pHER2, puPAR and pHu was transfected to breast cancer cells respectively, which all could inhibit cells proliferation, promote apoptosis, reduce cell invasiveness, inhibit activation of MAPK signaling pathway and make cells G0/G1 arrest. But pHu has a stronger efficiency than pHER2 or puPAR.(4) Breast cancer cells were treated with Herceptin, puPAR or both Herceptin and puPAR respectively. Then, breast cancer cells proliferation, apoptosis, cycles, invasiveness, and regulation of MAPK signaling pathway activation were detected and analyzed. The results showed that breast cancer cells were treated with Herceptin, puPAR or both Herceptin and puPAR respectively, which all could inhibit cells proliferation, promote apoptosis, reduce cell invasiveness, inhibit activation of MAPK signaling pathway and make cells G0/G1 arrest. But when breast cancer cells were treated with both Herceptin and puPAR has a stronger efficiency than Herceptin or puPAR alone.(5) Tumor-bearing mice were treated with Herceptin, puPAR or both Herceptin and puPAR, respectively. Then the Anti-tumor effect was detected and analyzed. The results of animal experiments showed that for HER2 low expression breast cancer cell line, puPAR has a strong effect to inhibit tumor growth, but Herceptin just has a low effect. When tumor-bearing mice were treated with both Herceptin and puPAR have a more significant addition or synergistic therapeutic effects than tumor-bearing mice were treated with Herceptin or puPAR alone.In conclusion: HER2 could regulate uPAR expression and uPAR also could regulate HER2 gene expression; Simultaneity inhibition HER2 and uPAR expression has a more significant addition or synergistic effects than inhibition HER2 or uPAR alone on inhibition cells proliferation, promotion apoptosis, decrease cell invasiveness, inhibition activation of MAPK signaling pathway and making cells G0/G1 arrest; Knocking down uPAR gene can increase the sensitivity of breast cancer cells to Herceptin.