The Role Of H Rad9 In Response To Chemotherapy And Its Value As A Therapeutic Target To Overcome Chemoresistance In Breast Cancer

Objective:hRad9/chkl signaling pathway plays an important role in DNA damage repair and is required for genomic stability.It was reported that hRad9 is over expression in many tumors and is correlated with tumor genesis and progress.However little is known about its role in response to chemotherapy of breast cancer. Our study is to investigate the role of hRad9 in the assessment of response to neoadjuvant chemotherapy and the feasibility of targeting it to overcome chemoresistance in breast cancer.Part Ⅰ The role of hRad9 in the assessment of response to chemotherapyMethods1.Fifty cases of invasive ductal breast carcinoma receiving neoadjuvant therapy (at least 4 periods) were collected from Qilu Hospital of Shandong University. All these cases’s Pre-neoadjuvant chemotherapy biopsy specimens and Post-neoadjuvant chemotherapy tumor specimens were collected and all its clinicopathological parameters were also completed.2.A11 H&E specimens before and after NAC were revised and estimated according to the Miller and Payne (MP) grading system.Specimens after NAC were classified into chemotherapy sensitive group(MP3-5) or chemotherapy resistant group(MP1-2).3.Immunohistochemistry were used to detect the hRad9,chkland CyclinDl expression in specimens before and after NAC.Results1.Positive expression rates of hRad9,chkl and Cyclin D1 is70%,42% and 50% respectively in specimens before NAC.2.A strong correlation was found between hRad9 over expression and chemoresistance. (χ2=40.9, P=0.000).3.A significant correlation was also found between chk1 over expression and impaired patient’s chemotherapy response (χ2=5.4, P=0.021)4.By bivariate analysis, a positive correlation was found between hRad9 and chkl (R=0.327, P=0.020,), hRad9 and Cyclin D1 (R=0.393, P=0.005).5.By Comparing the chemotherapy reactivity of hRad9 between Post-neoadjuvant chemotherapy tumor specimen and Pre-neoadjuvant chemotherapy biopsy specimen, we did not find significant variation for hRad9 expression before and after chemotherapy (P=0.571). Interestingly, chkl protein was significantly increased after neoadjuvant chemotherapy comparing with that of the biopsy specimen (P=0.012).Conclusions1. Over expression of hRad9 and chkl is correlated with chemotherapy resistance in breast cancer.2. hRad9 expression has a positive correlation with chk1 expression that suggests chkl/hRad9 plays an important role in chemoresistance.3. Chkl protein was significantly increased after neoadjuvant chemotherapy comparing with that of the biopsy specimen, while no significant variation for hRad9 expression before and after chemotherapy. It suggests that chkl might play an important role on acquired resistance, while hRad9 protein might contribute to the innate resistance of tumor cells response to chemotherapy.Part II Targeting hRad9 expression to overcome chemoresistance in breast cancerMethods1.Small interference targeted hRad9 and non-targeting siRNAs (as negative controls) sequences was designed and synthesized.2.Cells were divided into 3 groups:hRad9-siRNA,Negtive Control and Vehicle Control. MCF-7 and MDA-MB-231 cells were transfected with the above siRNAs respectively.3.RT-qPCR were used to determine the hRad9 mRNA expression.Cell immunohistochemistry and Western blot were used to detected the hRad9 protein expression in breast cancer cells.4. RT-qPCR and Western-blot were used to detect chkl mRNA and protein expression respectively in breast cancer cells.5.MTS assay was used to explore whether knockdown of hRad9 can increase sensitivity to doxorubicin of breast cancer cells.6.Cell apoptosis assay was used to explore the influence of knockdown of hRad9 on cell apoptosis in breast cancer.Results1. RT-qPCR showed hRad9-siRNA significantly decreased the expression of hRad9 mRNA compared with control cells. Cell IHC and Western blot analysis showed hRad9 protein expression was dramatically decreased in silenced versus non-silenced cells.2. RT-qPCR and Western blot analysis showed chkl mRNA and protein expression decreased in hRad9-siRNA cells compared to control cells.3. MTS analysis showed knockdown of hRad9 enhanced breast cancer cells sensitivity to doxorubicin.4. Flow cytometry analysis showed knockdown of hRad9 increased cell apoptosis in MDA-MB-231 breast cancer cells both before and after doxorubicin treatment.Conclusions1. Knockdown of hRad9 expression can potentiate the cytotoxic effects of chemotherapy and cell apoptosis on breast cancer cells that suggests hRad9 might be a potential predictor for the response to chemotherapy in patients with breast cancer.2. hRad9 can increase the chemotherapy sensitivity by regulating its down regulator chkl expression.However the role of chkl/hRad9 in chemoresistance remains to be further explored.