Down‐regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer

Abstract Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non‐muscle‐invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down‐regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p‐AMPK and p‐STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan‐Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down‐regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti‐bladder cancer activities. On the other hand, down‐regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 μmol L−1, 50 μL), the combination with metformin (60 mmol L−1, 50 μL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down‐regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.

immunosuppressive agents. 3,4 No substantial progress over the past 3 decades has been reported in clinical administration for bladder cancer except FDA-approved PD-L1 treatment in 2016. 5 Pirarubicin (THP, C32H37NO12) has a favourable antitumour efficiency with limited side effects; the potential application of THP in solid tumours has aroused great interest among researchers. Currently, it is one of the major drugs used in non-muscle-invasive bladder cancer instillation chemotherapy to improve chemotherapeutic efficacy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. 6 Furthermore, molecular mechanisms underlying this low response ratio are still not clear. Several scientists have demonstrated that when STAT3-specific siRNA knock-downs STAT3 expression or inhibitor (ie Stattic) inhibits STAT3 activity, it decreases the IC50 of THP treatment dramatically in bladder cancer cells. 7 This indicates that STAT3 plays an important role in the pharmacodynamics of THP. STAT3 is a member of the STAT family of transcription factors and is activated in several cancers including osteosarcoma, 8 renal cell carcinoma, 9 and pancreatic cancer. 10 Inhibition of STAT3 could serve as a potent chemotherapeutic sensitizing strategy to overcome drug resistance in chemoresistant cancer cells. 11,12 However, STAT3 is not an ideal molecular target for cancer therapy, because of its potential damage to normal tissue and other off-target effects. Gao et al 13 showed that PKM2 regulates constitutive activation of STAT3 in CRC cells. In this study, we are the first to observe that the expression of PKM2 in bladder cancer cell lines UMUC3, T24 and MB49 is inversely correlated with the IC50 values of THP treatment. This novel finding indicates that regulating PKM2 expression may be a valid strategy to enhance the efficiency of THP administration. Pyruvate kinase (PK) regulates the final rate-limiting step of glycolysis and catalyses the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate. 14 This transfer yields one molecule each of pyruvate and adenosine triphosphate. 15,16 PKM1, PKM2, PKL and PKR are PK isoforms expressed in different types of mammalian cells and tissues. Alternate splicing of PKM pre-mRNA by hnRNP A1/2 and polypyrimidine-tract binding (PTB) protein splicing factors leads to PKM2 generation by including exon 10 and excluding exon 9. [17][18][19] PKM2 has been an attractive target for cancer therapy because of its high levels of expression in most carcinomas. 20 Recent research strongly suggests that PKM2 plays an important role in the production of low-grade non-invasive and high-grade invasive urothelial carcinomas. 21 According to recent findings, high expression of PKM2 was associated with chemosensitivity to epirubicin and 5fluorouracil in breast cancer and also enhanced chemosensitivity to cisplatin in cervical cancer. 22,23 PKM2 has also shown contribution to gefitinib resistance via up-regulation of STAT3 activation in colorectal cancer. 24 Depletion of PKM2 led to apoptosis or sensitivity of several tumour cells to chemotherapy. 25,26 However, whether PKM2-induced STAT3 phosphorylation plays a significant role in the regulation of THP sensitivity in bladder cancer cells remains unclear.
PKM2 is closely related to metabolism. Our previous study has demonstrated that activated AMPK increases the efficacy of gefitinib on bladder cancer cells, related to metabolism-associated Warburg effect. 27 Furthermore, Li et al 28 showed that PKM2 inhibitor reverses AMPK activity. Thus, in this study, we aim to explore anticancer effect of THP and its molecular mechanisms on bladder cancers after downregulating PKM2, focusing on the roles of p-STAT3 and p-AMPK.

| Reagents
THP and Compound C were purchased from Selleck (Houston, TX, USA). Metformin was purchased from Aladdin chemistry Co. Ltd (Houston, TX, USA). They were diluted across a range of concentrations in culture media. Antibodies against PKM2, phosphor-STAT3(Y705), total STAT3, phosphor-AMPKa, total AMPKa and b-actin were purchased from Cell Signaling Technology (Cell Signaling, Beverly, MA, USA).

| Cell lines and culture conditions
Murine and human bladder cancer cell lines provided by Dr. P Guo were cultured in DMEM supplemented (Hyclone, Logan, UT, USA) with 10% of FBS (Hyclone, Logan, UT, USA) and 1% of penicillinstreptomycin at 37°C, in humidified air containing 5% of CO 2 .

| Human urothelial carcinoma tissues
Adjacent normal tissues (n = 6) and urothelial carcinoma tissues (n = 10) from patients who underwent transurethral resection or radical cystectomy of urothelial carcinoma were collected, following a protocol approved by both Institutional Review Boards of Xiangya Third Hospital, Central South University and Hunan Normal University. De-identified fresh tissues were fixation, paraffin-embedding and sectioning for immunohistochemistry. For scoring of immunohistochemical staining of human specimens, both the proportion and the intensity of the positive staining were scored following published methods, 29 with the proportion graded in 6 scales (0-5; ie, 0, none; 1,<1/100; 2, 1/100 to 1/10; 3, 1/10 to 1/3; 4, 1/3 to 2/3; and 5, more than 2/3), and the intensity graded in 4 scales (0-3; 0, none; 1, weak; 2, intermediate; and 3, strong). The total score from 0 to 8 was computed by combining the proportion and the intensity scores.

| Cell viability and cologenic assay
Cell viability was assessed using a tetrazolium-based assay using microplate reader (Biotek, SYNERGY HTX, Vermont, USA). IC 50 values were determined through the dose-response curves.
Cologenic survival was defined as the ability of the cells to form colonies. Images were taken and analysed by microscopy (Leica, DFC450C; Wetzlar, Germany) and microplate reader (Biotek, SYNERGY HTX).  Images were taken by microscopy (Leica, DFC450C) and semi-quantitative analysis with Image J; the average optical density indicates the fluorescence intensity; and the higher the average optical density, the more the number of apoptotic cells.   After centrifugation at 14 0009g for 15 minutes, the supernatant was collected.

| Animals
Female C57BL/6 mice were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China). Animals were housed 4 per cage in a specific pathogen-free animal facility. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Hunan Normal University.

| Orthotopic implantation and intravesical treatment
Exponential growth of MB49 cells 30 was harvested, and cell density in collection tube was counted by cell counter. Female mice 6 to 8 weeks of age were used for cancer cell implantation. Briefly, 1.2 9 10 5 MB-49 cells in 0.1 mL PBS were injected into the bladder wall using 1-mL syringes and catheter scratching according to the previously described protocol. 31 A total of 5 groups designed are Normal Female C57BL/6 mice which received intravesically an instillation of 50 lL of PBS as control group, Female C57BL/6 mice with orthotopic bladder cancer were randomly divided into 4 groups, which received intravesically an instillation of 50 lL of PBS metformin (60 mmol L À1 , 50 lL, twice per week), THP (400 lmol L À1 , 50 lL, twice per week), or combinations of metformin with THP-treated group (metformin 60 mmol L À1 , THP 400 lmol L À1 , total 50 lL, twice per week). Each group has 12 Female C57BL/6 mice. All treatments started at day 2 post-tumour implantation for 2 weeks. Lei Xue. The pathology evaluation was performed to confirm the presence or absence of tumour. Analysis of images was carried out using Image Pro Plus 4.5 software. The mean density was used as a semi-quantitative measure of relative immunoreactivity.

| Statistical analysis
All data are presented as mean AE SD. Statistical analyses were carried out using ANOVA, and statistical significance was assumed at a value of P < .05.

| PKM2 is highly expressed in bladder tumour tissue and tumour cells
We surveyed several established cell lines and found that PKM2 was highly expressed in human bladder cancer cell lines than primary-cultured human normal urothelial cell PEBC ( Figure 1A). As shown in Figure 1B,C, PKM2 was undetectable in normal urothelia tissues but highly expressed in urothelial carcinoma tissues by immunohistochemistry. These results are consistent with another observation that PKM2 is closely related to the development of bladder cancer. 21 3.2 | THP inhibits bladder cancer cell proliferation via activating AMPK, and this inhibitory effect is closely related to PKM2 Two human bladder cell lines were exposed to 0-640 nmol L À1 THP. THP exhibited a dose-dependent inhibition of cell proliferation.
The IC50 values were 146.80 and 247.24 nmol L À1 in T24 and UMUC3, respectively ( Figure 2A, Table 1). We next examined colony formation and found that THP exhibits a dose-dependent inhibitory effect at concentration range from 0 to 40 nmol L À1 ( Figure 2B). THP had a stronger inhibitory effect on T24 than on UMUC3 with similar pattern observed in proliferation assay described above. In order to clarify why different bladder cancer cells had different sensitivities to THP, we examined the PKM2 protein levels among the two cell lines after treatment with THP. The results revealed that THP reduced PKM2 expression in T24 and increased that of UMUC3. This novel finding implies that PKM2 is a key regulator to THP efficacy. Simultaneously, we discovered that AMPK was activated after THP treatment in UMUC3 ( Figure 2C) and T24 (Figure 2D). In order to further determine whether the inhibition of p-AMPK is associated with the reduced efficacy of THP, we combined Compound C, an inhibitor of p-AMPK with THP, and found a significant antagonistic effect in both UMUC3 ( Figure 2E) and T24 (Figure 2F) via MTT assay. In addition, UMUC3 cell lines treated by THP and Compound C also showed antagonism in cloning experiments ( Figure 2G). After treatment by Compound C in different F I G U R E 1 PKM2 is highly expressed in bladder tumour tissue and tumour cells. A, PKM2 expression was quantitated in 3 human bladder cancer cells and one normal bladder cell using WB; B, the expression of PKM2 in tissues was evaluated using immunohistochemistry. Two groups of specimens (N = 6 and 10 for adjacent normal tissues and cancer tissues, respectively) were paraffin-embedded, sectioned and immunohistochemically stained with anti-PKM2. Haematoxylin and eosin and Ki-67 were used to indicate whether there is a tumour. C, The differences between normal and tumour tissues regarding the expression levels of PKM2 were statistically significant (*P < .05, # P < .01) SU ET AL.

| Metabolism modulator-metformin inhibited PKM2
Metformin, a widely prescribed drug for treating type II diabetes, is one of the most extensively recognized metabolic modulators, with activating AMPK. 32 Metformin showed inhibitory effect in various cancer cell lines and xenograft cancer models and sensitized chemotherapy drugs. 33 Our recent study has demonstrated that metformin alone inhibited bladder tumour growth and increased the efficacy of gefitinib via activating AMPK. 34 To determine the role of metabolic modulators, we observed that metformin inhibited PKM2 expression obviously ( Figure 4A-B). PKM2 has monomer, dimer and tetramer forms under cross-linking conditions. 34 Dimer is active form of PKM2, and tetramer is inactive one. 35 Using cross-linker DTSSP, we are able to detect dimer and tetramer at pH 8.6 and pH 7.

| THP and metformin synergistically inhibited bladder cancer growth in vitro
Generally, at the range of tested concentrations (0-80 nM), compared with THP treatment alone, combination of THP with metformin exhibited a synergistic effect as assessed by CompuSyn software (Fig 5A). Colony formation assay showed that the inhibitory effect of THP increased when combined with metformin ( Figure 5B).

Cellular apoptosis was considerably increased when combined
with metformin at 24 hours detected by fluorescence microscopy ( Figure 5C).
Taken together, these results have demonstrated that the combined use of THP and metformin synergistically inhibited proliferation and colony formation of bladder cancer cells.

| Treatment of THP alone or combined with metformin on murine bladder cancer cell MB49
Since MB49 is the most popularly used murine bladder cancer cell line to establish syngeneic orthotopic bladder cancer model, we examined the inhibitory effect of THP alone or combined with metformin in MB49. As shown in Figure 6A, the IC50 value of THP in MB49 is 140.07 nmol L À1 . We also examined colony formation at concentration range from 0 to 40 nmol L À1 . It was found that THP exhibits a dose-dependent inhibitory effect in MB49 ( Figure 6B). We in UMUC3 and T24 ( Figure 6E-G). Furthermore, an intermittent treatment to MB49 with THP alone or combined with metformin for 2 hours was used to mimic intravesical treatment. Cells were treated twice per week for 2 weeks in vitro. The results demonstrated that combination of THP with metformin significantly reduced colony formation in MB49 at the indicated concentrations ( Figure 6H).
F I G U R E 5 THP and metformin synergistically inhibited bladder cancer growth in vitro. A, THP and metformin synergistically inhibited bladder cancer growth determined by MTT. Below: combination index (CI) among the combinations of 2 drugs was calculated using CompuSyn software. If CI = 1, it denotes additives; if CI >1, it denotes antagonism; if CI <1, it denotes synergism. CI values in the vast majority of combinations were less than 1, indicating synergism. Results are presented as the median of 5 independent experiments. B, T24 and UMUC3 were treated by Met(1 mmol L À1 ) and THP (20 nmol L À1 in UMUC3 and 10 mmol L À1 in T24) alone or combination; cell numbers were measured by colony formation assay after 7 days incubation. C, Met (10 mmol L À1 ) and THP (160 nmol L À1 ) alone or combination treated T24 and UMUC3 to indicate cell apoptosis via immunofluorescence experiments. Results are presented as the median of 5 independent experiments (*P < .05, # P < .01) A to determine the effect of intravesical localized treatment. 31 Syngeneic tumour implantation provided better tumour take rate compared to the xenograft implantation. 36 We reached 100% tumour take rate using previously reported technique. 31 The antitumour effect of THP was evaluated after combination with metformin using an orthotopic bladder cancer model in mice.  Figure 7B, the bladder weights in normal mice group were bigger than those in other groups. Intravesical treatment with THP or metformin alone profoundly diminished bladder weights.
The bladder weights between combination of metformin and THP group with normal group have no significant differences (combination treatment group vs normal mice group P = .078). Figure 7C

| DISCUSSION
Bladder cancer remains one of the main malignancies that affect the genitourinary tract. 37,38 The main clinical treatment is surgical resection assisted by chemotherapy. In fact, the unsolved severe problem of treating this disease is chemoresistance after surgical resection.
THP is one of the major clinically used drugs to treat bladder cancer via instillation administration. However, low response rate and side effect largely limit its efficacy.  40 This may be an efficient option to overcome the resistance and side effects of THP.
AMPK is activated allosterically by an increase in the intracellular AMP/ATP ratio. The most studied aspect of AMPK function is its role in maintaining cellular energy stores and regulating whole-body energy balance. 41 In the present study, we are the first to demonstrate that THP activated AMPK and the activation of AMPK is essential for THP to exert anti-bladder cancer. At the same time, we also find that AMPK is significantly activated after knocking down either PKM2 or STAT3. We confirm that activating AMPK signalling pathway is involved in down-regulating PKM2 to exert antitumour effect.
We also reveal that metformin, an AMPK activator, reduces the

CONFLI CT OF INTEREST
The authors declare no commercial or financial conflict of interest.