Osterix Reduces Chemosensitivity Of Breast Cancer Cells By Upregulating GALNT14 Expression

Breast cancer is one of the most common malignancies in women.In recent years,there is a gradually increasing incidence of breast cancer.Doxorubicin(dox)and paclitaxel(pax)are commonly used as the first-line chemotherapy drugs,but how to improve the sensitivity to these two kinds of drugs is still the main clinical problems to be addressed.Osterix(Osx),a key regulator of osteoblast differentiation and bone formation,plays a key role in osteoblast differentiation.Recent studies have shown that Osx is closely related to the metastasis of breast cancer,but the precise roles and mechanisms of Osx in breast cancer remains unclear.In our previous studies,we have constructed MDA-MB-231 stable cell lines with Osx overexpression or knockdown,as well as MCF-7 stable cell lines with Osx overexpression.In this study,using MTT assays we found that overexpression of Osx led to the decrease of the chemosensitivity of breast cancer cells to dox and pax,whereas knockdown of Osx led to the increase of the chemosensitivity of breast cancer cells.To further explore the underlying mechanisms by which Osx affects the chemosensitivity of breast cancer cells,we screened the differentially expressed mRNA between 231-OE6 and 231-OEC cells using high-throughput sequencing.Totally,47 up-regulated genes and 74 down-regulated genes were obtained.GALNT14 is one of the up-regulated genes with higher differential folds.GALNAT14 is a member of the N-acetylgalactosaminyltransferase family and studies have shown that decreasing GALNT14 expression could significantly enhance the chemosensitivity of hepatocellular carcinoma.Therefore,we hypothesized that Osx may affect the chemosensitivity of breast cancer cells by modulating the expression of GALNT14.To test this hypothesis,we constructed the expression plasmid of GALNT14,and synthesized specific siRNA of GALNT14.MTT assays showed that overexpression or knockdown of GALNT14 could reverse the increase or decrease in the chemosensitivity of breast cancer cells caused by Osx knockdown or overexpression.To elucidate the mechanisms by which Osx up-regulate the expression of GALNT14,we constructed the reporter plasmids containing the sequence of GALNT14 promoter with the potential binding site of Osx or not.The results showed that Osx had no significant effect on the promoter activity of GALNT14.It has been reported that GALNT14 played an important role in IGFBP-3-induced apoptosis and that GALNT14 was associated with tumor necrosis factor-related apoptosis-inducing ligand Apo2L/TRAIL signaling pathway.We speculated that Osx may inhibit the apoptosis of breast cancer cells by upregulating the expression of GALNT14,thereby reducing the chemosensitivity of breast cancer cells.By Hoechst staining and western blot assays,we found that Osx overexpression significantly upregulated Bcl-2 and downregulated the expression of Bax,and decreased the apoptosis of breast cancer cells,vice versa.At the same time,GALNT14 knockdown eliminated the inhibition of apoptosis caused by Osx overexpression,and GALNT14 overexpression obviously decreased the apoptosis induced by Osx knockdown.In addition,we constructed in situ breast cancer model in nude mice using MDA-MB-231 stable cell lines with Osx overexpression or knockdown and detected the expression of Osx and GALNT14 by immunohistochemistry(IHC).The results showed that the expression of GALNT14 was up-regulated in tumors established with Osx overexpression cells,and the expression of GALNT14 was down-regulated in tumors established with Osx knockdown cells.Moreover,we detected the expression of Osx and GALNT14 in 115 cases of breast cancer tissue using IHC analysis.Results showed that,of the 115 patients,89 patients exhibited higher expression of Osx(77.4%),58 of them exhibited higher expression of both Osx and GALNT14.26 patients presented lower expression of Osx,20 of them presented lower expression of both Osx and GALNT14.The results of SPSS analysis showed that there was a significant positive correlation between Osx and GALNT14 expression in breast cancer(p<0.01).Finally,we examined the expression of GALNT14 using breast cancer tissue microarray with 129 cases.The results showed that GALNT14 was highly expressed in patients with higher clinical stage,and the survival time was longer in patients with lower expression of GALNT14.Meanwhile,the expression level of GALNT14 was positively correlated with HER2 status(P=0.038),but not correlated with ER and PR expression.Taken together,we found that:? Osx reduced the sensitivity of breast cancer cells to dox and pac;? GALNT14 was involved in the regulation of chemo sensitivity by Osx;? Osx reduced the chemosensitivity of breast cancer cells by upregulating GALNT14,and then inhibiting the apoptosis of breast cancer cells;?The expression of Osx and GALNT14 in breast cancer tissues was significantly correlated,and GALNT14 may be used as a biomarker to predict the prognosis of patients with breast cancer.Results of this study indicates that Osx may be a novel target for drug design to improve the chemosensitivity of breast cancer.