Overexpression of HPIP as a biomarker for metastasis and prognosis prediction in endometrial cancer patients

Abstract Background Hematopoietic pre‐B‐cell leukemia transcription factor (PBX)‐interacting protein (HPIP) has shown to be overexpressed in several human cancers. The purpose of this study was to explore the expression of HPIP in endometrial cancer (EC) and its associated effects on disease. Methods A total of 113 EC patients at the Harbin Medical University Cancer Hospital between August 2011 and September 2012 were studied for immunohistochemistry analysis. HPIP expression was detected using real‐time reverse transcription PCR, Western blotting, and immunohistochemistry. Prognostic value of HPIP expression was examined using multivariate Cox regression analysis and Kaplan‐Meier method. Results The result of Western blotting indicated that HPIP protein expression is significantly high in normal tissues compared to EC tissues (P < 0.001). The expression of HPIP was significantly associated with FIGO stage (P < 0.001), histological grade (P < 0.001), depth of myometrial invasion (P < 0.001), and lymph node metastasis (P = 0.033). Kaplan‐Meier analysis demonstrated that there was a significant difference in overall survival and disease‐free survival between the two groups of patients stratified by HPIP expression level (log‐rank, both P = 0.002). Patients with HPIP high expression had significantly shorter median survival time than those with HPIP low expression. Moreover, results of the multivariate analysis revealed that HPIP expression was an independent prognostic factor for predicting overall survival (P = 0.015) and disease‐free survival (P = 0.017) in patients with EC. Conclusion The present study provides evidence that HPIP predicts EC progression and poor survival, highlighting its potential as a therapeutic target for EC.


| INTRODUC TI ON
Endometrial cancer (EC) is one of the most common cancers in women worldwide. 1 According to reports in the literature, the incidence of endometrial cancer in developed countries is higher than in developing countries. However, more and more women in China have been diagnosed with endometrial cancer in recent years. 2,3 Despite advances in the standard treatment for endometrial cancer, the prognosis in most patients with advanced endometrial cancer remains unsatisfactory. Therefore, it is imperative to further explore the occurrence and development of endometrial cancer progression and to identify therapeutic targets.
Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) was originally identified by a yeast two-hybrid screening of a hematopoietic cDNA library. According to the currently reported studies, HPIP exhibited an excessive expression status in numerous cancer tissues, while HPIP expression is involved in various aspects of cancer progression and predicts poor prognosis in cancer patients. [4][5][6][7][8][9][10][11][12][13][14][15] However, by reviewing the literature, we have not found a report on the expression of HPIP in endometrial cancer. Therefore, our research aimed to explore the expression status of HPIP in endometrial cancer and its relationship with clinicopathological features. At the same time, further evidence was provided for the possibility of HPIP as a potential target for the treatment of endometrial cancer.

| Patient population
This study has been obtained authorization from the Ethical Committee of the Harbin Medical University Cancer Hospital.
Patients of this study have signed written informed consent. In total, 113 endometrial cancer tissues were obtained from the Harbin Medical University Cancer Hospital. All patients received no treatment before surgery, and the cancer tissue samples were from patients who underwent gynecological surgery between August 2011 and September 2012. All patients underwent hysterectomy, bilateral salpingo-oophorectomy, pelvic and/or para-aortic lymphadenectomy, partial oophorectomy, and peritoneal lavage for cytology.
The follow-up ranged from 4 to 74 months, with a median followup of 62 months. All clinical information to be studied is summarized and listed in Table 1.

| Western blot analysis
Nine samples were frozen in liquid nitrogen. The lysate was mixed proportionally with a protease inhibitor and used to cleave the extracted protein. The BCA method was then used to determine the protein concentration in the sample solution. The next step is electrophoresis separation, membrane transfer, and closure.
After that, the primary antibody (anti-HPIP, 1:300, Abcam, LLC) hybridization and the secondary antibody (anti-β-actin, Santa Cruz Biotechnology) hybridization were carried out, and the membrane was removed for exposure identification. This experiment is repeated three times.

| Immunohistochemistry and evaluation
Detection of HPIP expression by immunohistochemistry. Paraffin specimens were sectioned for immunohistochemistry experiments. Then, dewax, rehydrate, and warm the bath. The antigen was repaired by heating with an autoclave and washed with PBS.  All immunohistochemical stains were scored by a professional pathologist who did not know the source of the sample.

| Real-time PCR
The expression of HPIP mRNA was quantified by RT-PCR. Nine samples were used for real-time PCR, including five endometrial cancer samples and four normal tissue samples. Total RNA was isolated using a TRIzol reagent (Wanlei) and converted to cDNA using the SuperScript III Platinum Kit (Invitrogen). Primers for HPIP are as follows: Forward, 5′-TTCTGGATGGCAGGAAGAT-3; Reverse, 5′-TCAAGGAGTCAAAGGAGGC-3. The primer sequence for β-actin as a reference is as follows: Forward, 5′-CGGGAAATCGTGCGTGAC-3; Reverse, 5′-GTCAGGCAGCTCGTAGCTCTT-3. Real-time PCR was performed with SYBR® Fast qPCR Mix (TaKaRa). Relative HPIP abundance was determined by the 2 −ΔC T method. This experiment was repeated three times.

| HPIP expression in patients with endometrial cancer
A total of 113 endometrial cancers samples were studied in our research for immunohistochemistry analysis. The clinicopathological features of the patients participating in the study are summarized in showed that the expression of HPIP was excessive at protein level in endometrial cancer samples (P < 0.001; Figure 1). Real-time PCR results showed that the expression of HPIP at mRNA level in endometrial cancer tissues is much higher than that in normal endometrial tissues (P < 0.05, Figure 2).

| HPIP expression is associated with clinicopathological feature in endometrial cancer
To further examine the expression pattern of HPIP in patients with endometrial carcinoma, HPIP expression was detected using F I G U R E 1 HPIP protein expression in normal and endometrial cancer tissues. A, Protein samples obtained from frozen normal endometrial tissues (N) and endometrial cancer tissues (T) were analyzed by Western blot analysis. Levels of β-actin were used as an internal control; B, histogram of pooled data from N (n = 4) and ECs (n = 5). HPIP expression was elevated in ECs compared with N. The data are presented as mean ± SD (***P < 0.001) F I G U R E 2 Histogram of HPIP mRNA expression in normal endometrial tissues (N) and endometrial cancer tissues (T). The levels of β-actin were used as an internal control, and the HPIP mRNA expression was calculated by 2 −ΔΔC t method. HPIP mRNA expression was elevated in CCs compared with normal endometrial tissues. The data are presented as mean ± SD (P < 0.05) immunohistochemistry. Results of immunohistochemical staining are shown in Figure 3A-D. As shown in Table 1 Excessive expression of HPIP was clearly associated with high FIGO stage (P < 0.001), deep myometrial invasion (P < 0.001), high histological grade (P < 0.001), and lymph node metastasis (P = 0.033; Table 1).

| Association between HPIP expression and prognosis of patients with endometrial cancer
According to HPIP expression, patients could be classified into two patient subgroups: patient group with high expression level of Results of Kaplan-Meier analysis suggested that there was significant difference in overall survival between the two groups of patients stratified by HPIP expression level (log-rank P = 0.002; Figure 4A). HPIP-overexpressed endometrial cancer patients had shorter median survival time (log-rank P = 0.002; Table 2). We further evaluated the capability of the HPIP expression in predicting disease-free survival. In consistent with the findings described above, the two groups of patients stratified by HPIP expression level showed significantly different disease-free survival (log-rank P = 0.002; Figure 4B). The disease-free survival of the HPIP overexpression group was clearly shorter (log-rank P = 0.002; Table 2).

| Independence of HPIP expression from other clinicopathological factors
To assess whether prognosis prediction ability of HPIP expression is independent of other clinicopathological features of patients with endometrial carcinoma, we performed multivariate Cox regression analysis. As shown in

| D ISCUSS I ON
In this study, we investigated whether HPIP is highly expressed in endometrial cancer tissues and its association with clinicopathological features. Furthermore, this study also explored the prog- endometrial cancer and it could be used to index the prognosis and metastasis of endometrial cancer.
As shown in Figures 1 and 2, the consequence of real-time PCR (mRNA levels) and Western blot (protein level) certified that excessive HPIP expression was found in EC samples nevertheless not found in normal samples. In this study, we performed data analysis on the results of immunohistochemistry of 113 endometrial cancer samples. Data analysis showed that overexpressed HPIP was highly correlated with clinicopathological features of endometrial cancer, including FIGO stage, myometrial invasion, histological grade, and lymph node metastasis. All of the above studies indicated that HPIP is involved in and plays a non-negligible element in promoting cancer progression. We found that patients with HPIP high expression had worse disease-free survival and overall survival through Kaplan-Meier analysis and log-rank analysis. As shown in Table 3  closely related to cancer progression and prognosis. This study provided strong support and evidence for HPIP as a new biomarker and the potential therapeutic target for endometrial cancer. But these findings require more extensive experiments to verify.

AUTH O R S ' CO NTR I B UTI O N S
HF and FLM conceived and designed the experiments; LC, TTZ, SGL, and YW performed the experiments and analyzed the data; and HF and FLM wrote the paper. All authors read and approved the final manuscript.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
This research was completed in compliance with the Helsinki Declaration. The data collection and analysis were carried out without disclosing patients' identities.