CVT-313 | Nur77 Signal

Molecular analysis of cell survival and death pathways in the proteasome inhibitor bortezomib‑resistant PC3 prostate cancer cell line

Ertan Kanbur1 · Ahmet Tarık Baykal2 · Azmi Yerlikaya3

Received: 1 July 2021 / Accepted: 2 August 2021 / Published online: 7 August 2021
© Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
The ubiquitin–proteasome pathway is an important protein quality control system involved in intracellular homeostasis. To achieve intracellular homeostasis, proteins that are misfolded as a result of translational errors or genetic mutations must be eliminated by the ubiquitin–proteasome pathway. In our previous publications, we determined that 4T1 breast and B16F10 melanoma cancer cells have differential levels of resistance to proteasome inhibitors. Again, in the previous studies, we reported that 4T1 cell cultures, despite being p53-mutant, underwent apoptosis as a result of bortezomib treatment. The first goal of this study was to verify the resistance levels of parental and resistant PC3 prostate cancer cells to bortezomib using WST-1 test. As a result of treatment with different bortezomib concentrations for 48 h, the IC50 value of the parental cells was determined as 32.8 nM and that of the resistant cells was determined as 346 nM. This result showed that the resistant cells were at least 10.5 times more resistant. In addition, to determine whether the resistance gained was reversible or not, the cells were passaged in a medium without bortezomib for one month. The IC50 value determination by WST-1 test showed that the resistant PC3 cells gained an irreversible bortezomib resistance phenotype. The results of the 3D spheroid experiment showed that the 3D spheroid diameter of resistant cells was significantly higher than that of the parental cells. The studies conducted with Western blot showed that ERK1 MAPK T202 phosphorylation and the conversion of autophagy marker LC3-I to LC3-II were significantly increased in parental cells as compared to the resistant cells. Finally, the results showed that while both maternal embryonic leucine zipper kinase (MELK) inhibitor OTSSP167 and Ca2+ chelator BAPTA-AM (also an inhibitor of the expression of antiapoptotic protein GRP78) are promising agents for cancer cells resistant to the proteasome inhibitors, CDK2 inhibitor CVT-313 was found ineffective in both parental and the resistant cells.
Keywords Bortezomib · Prostate · Cancer · ERK · LC3 · CDK2 · MELK

Abbreviations
AML Acute myeloid leukemia
MELK Maternal embryonic leucine zipper kinase CDK2 Cyclin-dependent kinase 2
HSP Heat shock protein FBS Fetal bovine serum
IC50 Inhibition concentration 50

 Azmi Yerlikaya [email protected]
1 Department of Immunology, Faculty of Medicine, Bursa Uludağ University, Bursa, Turkey
2 Department of Medical Biochemistry, Faculty of Medicine, Acıbadem Mehmet Ali Aydınlar University, Istanbul, Turkey
3 Department of Medical Biology, Faculty of Medicine, Kutahya Health Sciences University, Kutahya, Turkey

ERK Extracellular signal-regulated kinase
LC3 Microtubule-associated proteins 1A/1B light chain 3B
PC3-P Parental PC3 PC3-R Resistant PC3

Introduction
The ubiquitin–proteasome pathway is the most important intracellular proteolytic system and its function is crucial to cellular homeostasis [1, 2]. Target proteins are often degraded by the 26S proteasome after labeling with the 76 amino acid ubiquitin molecule. The binding of ubiquitin to target proteins is covalent and triggers ATP-dependent degradation [3]. It has been shown that proteasome sub- units or activity increase in many types of cancer (e.g.,

colon, prostate, melanoma, kidney, lung, and liver) [4–6]. For example, compared with healthy individuals, it has been shown that the amount of 20S proteasome in acute myeloid leukemia (AML) and Hodgkin’s disease plasma increases 2- to 12-fold [7]. These results show that the ubiquitin–proteasome pathway plays an important role in cancer development. For these reasons, intensive studies are being conducted on new inhibitors or combined thera- pies that specifically target the ubiquitin–proteasome path- way. Targeting the ubiquitin–proteasome pathway using proteasome inhibitors is therefore seen as a new approach for cancer therapy as well as to overcome resistance mechanisms developed during the treatment. Compared to normal cells, tumor cells, especially myeloma cells, are more sensitive to the proteasome inhibitors [8]. Clini- cally, proteasome inhibitors have yielded positive results in the treatment of multiple myeloma, non-Hodkin’s lym- phoma, mantle cell lymphoma, non-small cell lung carci- noma, prostate cancer, pancreatic cancer, and colon cancer [9–13]. However, the expected clinical activity could not be achieved in patients with fludarabine-refractory CLL, treated with proteasome inhibitor bortezomib, which is assumed to be due to the short serum half-life and/or to its reversible inhibition of the proteasome [14]. Similarly, Markovic et al. reported that single-agent bortezomib, administered twice weekly × 2 weeks, every 3 weeks at a dose of 1.5 mg/m2, was not found to be effective in the treatment of patients with metastatic melanoma [15]. Therefore, more studies should be carried out on the effects of inhibition of the ubiquitin–proteasome pathway on different types of cancer.
Bortezomib (also known as Velcade or PS-341) reversibly inhibits the chymotrypsin-like activity of the proteasome. Chemically, it is a dipeptidyl boronic acid analog derived from leucine and phenylalanine. Bortezomib has been shown to inhibit tumor cell proliferation, adhesion, metastasis in many in vivo and in vitro models, and has been approved by the US Food and Drug Administration (FDA) for use in cancer treatment [9, 16]. Proteasome inhibitors trigger apoptosis in a p53-dependent manner in many cell cultures such as Rat1 fibroblast, PC12 pheochromocytoma, HCII and KIM-2 mammary epithelial cells [17, 18]. However, in some other cells (prostate, HCT 116 colon cancer, neuroblastoma, or lymphoma), it is also known that proteasome inhibition induces apoptosis in a p53-independent manner [19, 20]. In our own studies, we determined that 4T1 breast cancer cells are p53-mutants and that proteasome inhibitors induce apoptosis as observed by DNA ladder analysis and caspase-3 activity [21, 22]. Our studies with Western blot showed that pro-apoptotic proteins such as Puma, Noxa, Bax, Bad, and caspase-7 are not critical regulators of apoptosis induced by the proteasome inhibitor bortezomib in p53-mutant 4T1 cells [22].

As with many chemotherapeutic agents, the most impor- tant conundrum to treatments with proteasome inhibitors is the development of either natural or acquired resistance mechanisms under the sustained drug pressure. To shed light on the molecular mechanisms of acquired resistance to proteasome inhibitors (particularly bortezomib), sev- eral bortezomib-resistant cell lines have been established by stepwise increasing concentrations of bortezomib. The results obtained include various mutations in proteasome subunit PSMB5, overexpression of proteasomal subunits, alterations in stress response, cell survival, and anti-apop- totic pathways, as well as changes in the expression of genes involved in multidrug resistance [23, 24]. In the study by Oerlemans et al., it has been shown that bortezomib resist- ance in the human myelomonocytic THP1 cell line is associ- ated with an Ala49Thr mutation in the PSMB5 protein, and there was also a dramatic increase in PSMB5 expression up to 60-fold. However, no change was observed in other proteasome subunits (PSMB6, PSMB7, and PSMA7) [25]. In another study, Wu et al. developed two hepatocellular cancer cell lines resistant to bortezomib, and although an increase in the expression of the PSMB1 and PSMB5 protea- some subunits was observed in these cells, no mutation was observed in the PSMB5 subunit to which bortezomib binds [26]. Lü et al. showed that bortezomib resistance was caused by the PSMB5 gene overexpressed in T-lymphoblastic lym- phoma/leukemia and that it was caused by a mutation in the PSMB5 gene (G322A) [27, 28]. Using PC3 prostate cancer cell line as a model system, we previously found that the precursor form of PSMB5 was overexpressed in response to bortezomib treatment in the parental cell, while there were no changes in its expression pattern or processing in the resistant cell line [29]. Heat shock proteins are over- expressed in many tumors in stressful environments and have been reported to be associated with a low prognosis and chemotherapeutic resistance [30]. Shringarpure et al. reported that heat shock proteins HSP70, HSP27, HSP90 are significantly expressed in bortezomib-resistant primary B lymphoma cells (SUDHL-4) than that in bortezomib- sensitive cells (SUDHL-6) [31]. These studies show that the resistance mechanisms developed against proteasome inhibitors should be investigated in more detail to develop new and more effective treatment protocols.
The current study aims to elaborate on the resistance
mechanisms of PC3 prostate cells against proteasome inhibi- tor bortezomib and to contribute to the development of a new alternative treatment protocol for prostate cancer treat- ment and other types of cancer. In line with this purpose, our specific goals are to investigate the effects of bortezomib on the 3D spheroid model in parental and resistant PC3 prostate cancer cells, as well as the activation of the cell survival pathway ERK1/2 MAPK pathway and the conversion of autophagic death marker LC3-I to LC3-II. Finally, we tested

the effects of MELK inhibitor OTSSP167, CDK2 inhibitor CVT-313, and Ca2+ chelator BAPTA-AM on the parental and resistant cell proliferation.

Materials and methods
Materials

DMEM media, 10X trypsin solution, penicillin/streptomy- cin, fetal bovine serum (FBS), RIPA buffer, HEPES, sodium pyruvate, sodium bicarbonate, sodium chloride, agarose, acrylamide, bis-acrylamide, D-(+)-glucose, sodium dode- cyl sulfate, TEMED, developer and replenisher, fixer and replenisher were purchased from Sigma-Aldrich. Protein Assay Dye and protein standard BSA were purchased from Bio-Rad. UltraCruz® Protease Inhibitor Cocktail was pur- chased from Santa Cruz Biotechnology. Cell proliferation reagent WST-1 was purchased from ROCHE Diagnostics. PC3 cell line (ATCC Cat# CRL-1435, RRID: CVCL_0035)
[32] was provided by Prof. Dr. Serap Kuruca Erdem (Istan- bul University, Istanbul, Turkey).
WST‑1 assay

The resistant PC3 cells were obtained as described before by a stepwise increase of bortezomib concentrations over six months [29]. The IC50 values of bortezomib in both paren- tal and resistant cells were determined by WST-1 assay as briefly described below. First, the parental PC3 and resist- ant PC3 cells (10,000) cells were seeded in each well of a 96-well plate. After 24 h of seeding, the cells were treated with different concentrations of bortezomib (1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 10 µM, 50 µM, 100 µM)
for 48 h at the logarithmic phase of growth (70% conflu- ency). The control groups were treated with an isotonic solu- tion. Subsequently, the medium was replaced with DMEM containing 0.5% FBS + 10 mg/ml WST-1 for 2 h at 37 °C. Afterward, the cell survival was determined by reading the absorbances of each well by RT-2100C microplate reader at 450 nm. The results were then analyzed with GraphPad Prism 5 program. The IC50 values of bortezomib were then obtained by using nonlinear regression to fit the data to the log(inhibitor) vs. response or log(inhibitor) vs. response- variable slope [33].
3D cell culture

The 3D spheroids of parental PC3 and resistant PC3 cells were generated as described previously [33]. Briefly, to prepare 1% agarose-coated 96-well plates, DMEM media containing 10% FBS was mixed with an appropriate amount of 10% agarose (dissolved in dH2O and sterilized at 121 °C

for 20 min) to obtain the desired concentration [1% (wt/ vol) in the cell culture medium]. After cell counting, 500 cells were seeded on top of the solidified DMEM contain- ing 1% agarose in a 100 μl total cell culture medium. The cells were incubated for three days before treatment. After- ward, an appropriate amount of bortezomib was added to each well to obtain the desired concentrations (20 nM bort- ezomib and 100 nM bortezomib). The control groups were treated with an isotonic solution. The spheroid morphology and diameter in each well was monitored and photographed every third day for 15 days by using an inverted microscope (AE21, Motic Europe) using 4X objective. The results were analyzed by GraphPad Prism 5 program. One-way ANOVA and Bonferroni’s multiple comparison tests were applied to determine the statistical significance. A p value of 0.05 or less was considered significant.
Western blot analyses

Western blot analysis was performed as described before [29]. Briefly, the parental PC3 and resistant PC3 cells (200,000/35 × 10 mm dishes) were grown to the exponential phase of the growth and then treated with 20 nM carfilzomib, 20 nM bortezomib, and 100 nM bortezomib or with an iso- tonic solution as a control group for 24 h. Cells were lysed with RIPA buffer (Cat# R0278, Sigma-Aldrich) containing 1X protease inhibitor cocktail (Cat# sc-29131, Santa Cruz Biotechnologies Inc.). The protein quantification was deter- mined by the Bradford assay [34]. Subsequently, 35 µg pro- tein from each sample was separated on a 12% (for ERK1/2 and LC3 analysis) or 10% (for HSP70 analysis) SDS-PAGE, followed by a transfer to a PVDF membrane (Cat# 1704156, Bio-Rad Laboratories) using Bio-Rad Trans-Blot Turbo Transfer system. The membranes were then probed with rabbit monoclonal anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody (1:1000 or 1:750 dilution, Cat# ab201015, Abcam). The membranes were stripped and re- probed with rabbit polyclonal anti-ERK1 + ERK2 antibody, recognizing the total form of the protein (1:1000, dilution, Cat# ab17942, Abcam). For the analysis of HSP70 expres- sion, the membrane was probed with a mouse monoclo- nal anti-HSP70 antibody (Cat# H5147, Sigma-Aldrich). Autophagy activity was detected by probing membranes with rabbit polyclonal anti-LC3-B antibody (1:1000 dilution, Cat# 2775, Cell Signaling Technology Inc.). Equal protein loading was determined with an anti-β-actin rabbit poly- clonal antibody (1:3000, Cat# ab8227, Abcam) in TBS-T for 1 h. After incubation with the primary antibody for 1 h, an anti-rabbit HRP-conjugated secondary antibody (1:3000, Cat# 7074, Cell Signaling Technology Inc.) or anti-mouse HRP-conjugated secondary antibody (1:3000, Cat# 7076, Cell Signaling Technology Inc.) was added to the membrane for 1 h in TBS-T. Finally, to visualize the protein bands,

membranes were incubated with LumiGLO reagent (Cat# 7072, Cell Signaling Technology Inc.), and the emitted light was captured on an X-ray film in a dark room.
Label free nLC‑MS/MS analyses

The experiment and analysis were carried out as described previously [35]. Briefly, after separation by %12 SDS-PAGE, proteins were stained with %0.1 coomassie blue for 1 h and washed 3X by a distaining solution until bands are visual- ized. Then, the bands were cut out for label-free nLC-MS/ MS analysis. The excised gel pieces were washed for 15 min in 500 µl 100 mM EDTA/NaOH on a shaker. The superna- tant was discarded and an appropriate amount of 50 mM dithiothreitol (prepared in 20 mM ammonium dihydrogen phosphate) was added to cover the gel pieces on a shaker at 55 °C for 10 min. The supernatant was discarded again and 200 mM iodoacetamide (prepared in 20 mM ammonium dihydrogen phosphate) was added to cover the gel pieces and incubated for 20 min in dark. The supernatant was discarded and 100% acetonitrile was added to dehydrate gel pieces for 5 min on a shaker. The supernatant was discarded again and 30% acetonitrile was added for the rehydration of gel pieces for 5 min on a shaker. The supernatant was discarded and the gel slices were washed 2× with 1 ml of ultra-pure H2O for 10 min on a shaker. After discarding the supernatant, the gel slices were washed with 1 ml of 50% acetonitrile + 20 mM ammonium dihydrogen phosphate 5× for 30 min on a shaker until all the dye residues were removed. The pep- tides were obtained by treatment with trypsin (dissolved in 50 mM ammonium bicarbonate containing 2% acetonitrile) at 37 °C for 16 h. The tryptic peptides were analyzed by Xevo G2-XS Q-TOF (Waters), the detector and calibrations were carried out with the MassLynx program (V4.1-Waters). The tryptic peptides were fractionated through the HSS T3 (Waters-186008818) columns using an acetonitrile gradient. During analyses, the data for the peptides within the m/z 50-1950 range were collected. Protein identifications were made using human protein sequences in the UniProt protein database. Progenesis QIP software (Waters-2018) was used for protein identification and statistical analyses.
iCELLigence system

The effects of MELK inhibitor OTSSP167 and CDK2 inhibitor CVT-313 as well as Ca2+ chelator BAPTA-AM on the proliferation of parental and resistant PC3 cells were determined by using the iCELLigence system as described in detail previously [36]. First, 20,000 cells were seeded in each well of E-plate L8 containing integrated microelectrode sensors in the bottom of each well. As the cells proliferate and adhere to the micro-electrodes, the changes in electri- cal impedance occur reflecting the proliferation rates of the

cells. The system therefore allowed real-time monitoring of the effects of inhibitors on the parental and resistant cells. The cell proliferation status was expressed in terms of cell index (CI). The inhibitors (100 nM OTSSP167, 100 nM CVT-313, and 20 µM BAPTA-AM) were added to the wells after 24 h of seeding in the logarithmic phase of the growth in 500 µl medium. The cells were then incubated at 37 °C, 5% CO2 for 96 h. The cell index (CI) was monitored every 30 min by the iCELLigence system.

Results
We previously developed a PC3 prostate cancer cell line resistant to the proteasome inhibitor bortezomib by stepwise increasing concentrations of bortezomib to uncover molecu- lar mechanisms leading to drug resistance [29]. We showed that multidrug resistance (MDR) transporters and overex- pression of the proteasomal subunit PSMB5 were not the critical regulators of the resistance. In the same publication, it was also reported that the cells were in part cross-resistant to carfilzomib, an irreversible inhibitor of the 26S protea- some [29]. Here, we aimed to elaborate on the molecular mechanisms of resistance to the proteasome inhibitor bort- ezomib. Initially, to confirm the resistance status of the cells, the IC50 values of both parental and resistant cells were re- examined by WST-1 assay after 48 h of treatment. As can be seen in . 1A and B, the IC50 value of parental PC3 cells was determined as 32.8 nM, whereas that of the resist- ant cells was 346 nM, indicating a 10.5-fold increase in the resistance. During the cell passages, the resistant cells were kept under 5 nM or 10 nM bortezomib pressure to avoid loss of the resistance phenotype for about ten more months. After that period, to determine whether the resistance was revers- ible or not, the resistant cells were then cultured without bortezomib for more than one month. As seen in Fig. 1C, while the IC50 value of the parental cells was similarly determined as 34.3 nM, that of the resistant cells was more than 100 µM, indicating that the cells gained an irrevers- ible resistance phenotype. Apparently, 5 nM or 10 nM bort- ezomib pressure on the resistant cells caused an elevated as well as an irreversible resistance status by selective survival of more resistant cells. Then, the response of both parental and resistant cells to 20 nM bortezomib treatment was deter- mined with a 3D spheroid formation model, which is much more representative of in vivo cell behaviors [37]. As shown in 2A and B, the resistant cells were able to form the 3D spheroids upon exposure to 20 nM bortezomib concentra- tion starting from day 6. However, the parental cell were not able to form any spheroids until day 15. Both parental and resistant PC3 cells were not able to form the 3D spheroids in the presence of 100 nM bortezomib ( 2A and B). As can be seen in . 2A and B, the 3D spheroid diameter

Fig. 1 Determination of the IC50 values. The IC50 values of the paren- tal (A) and bortezomib-resistant cells (B) were determined with vary- ing concentrations of bortezomib after 48 h of treatment as described in the Material and Methods section using WST-1 test. C To deter- mine the reversible nature of resistance, the resistant cells were cul-

tured in the absence of bortezomib pressure for more than one month, and then the IC50 values were determined similarly after 48 h treat- ment of the varying concentration of bortezomib. The results are pre- sented as means ± SEM (n = 3)

the parental control cells was much bigger than that of the resistant control cells, which is probably due to the higher proliferation rates as reported in our previous study [29]. Therefore, we next examined the expression and phospho- rylation status of the extracellular signal-related kinases (ERKs), activating pro-survival pathways and modulating apoptosis, differentiation, senescence as well as chemore- sistance in tumor cells [38]. In addition, Wang et al. inter- estingly showed that ERK-mediated autophagy can lead to cisplatin resistance and suggested that cisplatin resistance can be overcome by knockdown of ERK or inhibition of autophagy in acquired cisplatin-resistant ovarian cancer cells [39]. We therefore initially examined the total ERK1/2 expression and phosphorylation status in response to 20 nM and 100 nM bortezomib as well as 20 nM carfilzomib treat- ments for 6 h and 24 h in both parental and resistant cells using Western blot analysis. As seen in 3A, there was no significant change in the expression level of total ERK1 and ERK2 after treatment with the indicated inhibitor concen- trations and time intervals. However, the findings indicated that ERK1 T202 phosphorylation was increased in a dose- dependent manner in the parental cells with about a 2.3-fold

increase after 100 nM bortezomib treatment as compared to the parental control cells ( 3A and B). Nonetheless, there was no significant change in ERK1 T202 phosphoryla- tion after 6 h of treatment with 20 nM and 100 nM bort- ezomib concentrations in the resistant cells (. 3A and B). While 20 nM carfilzomib caused a decrease in ERK1 T202 phosphorylation in the parental cells, about 50% increase in ERK1 T202 phosphorylation was observed in the resistant cells after 20 nM carfilzomib treatment (. 3A and B). On the other hand, there was no significant change in the level of ERK2 T185 phosphorylation between the parental and resistant cells after 6 h of bortezomib or carfilzomib treat- ments ( 3A and C). Interestingly, after 24 h of treatment with 100 nM bortezomib, ERK1 T202 phosphorylation was increased about 25-fold in the parental cells as compared to a fivefold increase in the resistant cells, indicating that ERK1 T202 phosphorylation is responsive to the degree of proteasomal inhibition (. 4A and B). This inference is pri- marily derived from the fact that the resistant cells are not as responsive as the parental cell to bortezomib treatment due to an acquired mutation in the proteasomal PSMB5 subunit, as inferred from their irreversible resistance mechanisms.

Fig. 2 The 3D spheroid formation. A The parental and resistant cells were treated with 20 nM or 100 nM bortezomib at day 3 after cell seeding. The experiment was ended on day
15. B The spheroid diameters were expressed as the % of control. Using GraphPad Prism 5 program, One-way ANOVA and Bonferroni’s multiple com- parison tests were applied to determine the statistical signifi- cance. ***represents p < 0.001. The results are presented as means ± SEM (n = 3)

Parental Con Parenal 20 nM Parental 100 nM Resistant Con Resistant 20 nM
Resistant 100 nM

Also, in our previous study, we reported that the polyubiq- uitinated conjugates were not increased significantly in the resistant cells as much as that observed in the parental cells in response to bortezomib treatment [29], again an indication of higher proteasomal activity gained through an acquired mutation in the proteasomal subunit, making the proteasome insensitive to the inhibition by bortezomib. Unexpectedly, the level of ERK2 T185 phosphorylation was decreased in both parental and resistant cells after treatment with 20 nM and 100 nM bortezomib ( 4A and C). However, as seen in Fig. 4A and C, a more significant reduction in ERK2 T185 phosphorylation was observed in the parental cells as com- pared to the resistant cells. Recent studies show that ERK activation is critical for regulating autophagic cell death. However, there are a number of controversial results whether ERK phosphorylation is indeed an inducer or an inhibitor of the autophagic flux [40, 41]. Several studies also indi- cated that autophagy activation is closely associated with

the resistance to chemotherapeutic agents [42, 43]. There- fore, we next examined the conversion of autophagic marker LC3-I to LC3-II (a phosphatidylethanolamine (PE) conju- gated form of LC3-I) in both parental and resistant cells in response to the proteasomal inhibition. As seen in Fig. 5, LC3-I is converted to LC3-II form in the parental cells in response to 20 nM and 100 nM bortezomib; whereas no conversion of LC3-I to LC3-II is observed in the resistant cells, indicating the activation of autophagic cell death in the parentals as a compensatory mechanism under the condi- tions of proteasomal inhibition. Interestingly, 20 nM carfil- zomib treatment did not affect the conversion of LC3-I to LC3-II in both cells ( 5). Since ERK1 T202 and ERK2 T185 phosphorylation levels are differentially regulated in response to the proteasomal inhibition in the parental cells, it is likely that ERK1 T202 phosphorylation may be critical for the conversion of LC3-I to LC3-II, and thus the autophagic activation. Following Western blot analysis, the

Fig. 3 Western blot analysis of ERK1 and ERK2 phosphoryla- tion after 6 h of treatment of the parental (PC3-P) and the resistant (PC3-R) cells. A 35 µg protein was separated on 12% SDS-PAGE fol- lowed by Western blot using rabbit monoclonal anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody or with rabbit polyclonal Anti- ERK1 + ERK2 antibody recognizing the total forms. The paren-

tal (PC3-P) and resistant (PC3-R) cells were treated with 20 nM and 100 nM bortezomib or 20 nM carfilzomib for 6 h. B) Quantitation of ERK1 (phospho T202) and ERK2 (phospho T185) phosphorylation levels after normalization using the total forms of the proteins. The results are representative of an experiment run in duplicate

SDS-PAGE gels are routinely stained with 0.1% coomassie blue to check the equal protein loading; however, as can be seen in . 6A, we surprisingly observed a high molecular weight protein, increasing with the higher bortezomib con- centrations as well as carfilzomib treatment in the parental cells without any change in the resistant cells. This protein band was excised and analyzed by label-free nLC-MS/ MS, and was found as representing HSP70 protein fam- ily members with high confidence (Table 1). Western blot analysis also proved that HSP70 protein was increased after treatment with bortezomib and carfilzomib in the parental cells
Recently, Zhang et al. showed that maternal embryonic leucine zipper kinase (MELK), a serine/threonine kinase, played an essential role in the chemoresistance of uterine leiomyosarcoma (ULMS) cells, the most lethal gyneco- logic malignancy [44]. Similarly, cyclin-dependent kinase 2 (CDK2) also promotes hyperproliferation and is associated with poor prognosis, chemoresistance and radioresistance mechanisms in multiple cancer cells [45, 46]. Accumulating evidence shows that the antiapoptotic and ER-resident pro- tein GRP78 also influences chemoresistance and prognosis in cancer [47]. In our previous studies, we also reported

that MELK substrate Drebrin-like (DBNL), CDK2 substrate Cdc5L, and GRP78 proteins were overexpressed in response to the proteasomal inhibition [33, 48]. Therefore, we next examined the effects of MELK inhibitor OTSSP167, CDK2 inhibitor CVT-313, and BAPTA-AM, a Ca2+ chelator as well as an inhibitor of GRP78 expression [49] on the parental and resistant cells. As seen in 7, while MELK inhibitor OTSSP167 (100 nM) and Ca2+ chelator and GRP78 inhibitor BAPTA-AM (20 µM) similarly inhibited the proliferation of the parental and resistant cells, CDK2 inhibitor CVT-313 (100 nM) was found ineffective in both cells using the real- time cell analysis system iCELLigence. The results showed that both OTSSP167 and BAPTA-AM inhibitors are promis- ing agents for proteasome inhibitor-resistant cancer cells.

Discussion
Chemoresistance is the major problem in cancer therapy, and in fact, over 90% mortality of cancer patients is attrib- uted to the development of drug resistance mechanisms [50, 51]. Therefore, the mechanisms underlying the resistance to chemotherapeutics and alternative treatment protocols need

Fig. 4 Western blot analysis of ERK1 and ERK2 phosphoryla- tion after 24 h of treatment of the parental (PC3-P) and the resistant (PC3-R) cells. A 35 µg protein was separated on 12% SDS-PAGE fol- lowed by Western blot using rabbit monoclonal anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody or with rabbit polyclonal Anti- ERK1 + ERK2 antibody recognizing the total forms. The paren-

tal (PC3-P) and resistant (PC3-R) cells were treated with 20 nM and 100 nM bortezomib or 20 nM carfilzomib for 24 h. B) Quantitation of ERK1 (phospho T202) and ERK2 (phospho T185) phosphorylation levels after normalization using the total forms of the proteins. The results are representative of an experiment run in duplicate

Fig. 5 Western blot analysis of LC3-I and LC3-II conversion after 24 h of treatment of the parental (PC3-P) and the resistant (PC3-R) cells. The autophagic activation was determined by examining the conversion of LC3-I to LC3-II using rabbit polyclonal anti-LC3- B antibody. Equal protein loading was determined by reprobing the

membranes with an anti-β-actin rabbit polyclonal antibody. The parental (PC3-P) and resistant (PC3-R) cells were treated with 20 nM and 100 nM bortezomib or 20 nM carfilzomib for 24 h. The results are representative of an experiment run in duplicate

to be investigated in detail and developed urgently for bet- ter clinical outcomes. Preclinical in vitro and in vivo stud- ies showed promising antitumor potential of bortezomib as a single agent against androgen-dependent (LNCaP) and androgen-independent (PC-3 and DU145) prostate cancer cell lines [52, 53]. However, clinical phase I and/or phase II studies indicated that administration of bortezomib as

a single agent has minimal antitumor activity in patients with advanced androgen-independent prostate cancer. On the other hand, the combination of bortezomib plus mitox- antrone showed potential antitumor activity in patients with advanced androgen-independent prostate cancer [54, 55]. Therefore, it is believed that the proteasome inhibitors will be included in the combination regimens against a number of

Fig. 6 A Visualization of high molecular weight proteins in the parental cells using coomassie blue staining. The parental (PC3-P) and the resistant (PC3-R) were treated with 20 nM and 100 nM borte- zomib or 20 nM carfilzomib for 24 h. Following Western blotting, the SDS-PAGE gels were stained with 0.1% coomassie blue and washed

extensively with a destaining solution. B Western blot analysis of HSP70 after the treatment of parental and resistant cells with 20 nM and 100 nM bortezomib or 20 nM carfilzomib for 24 h. The results are representative of three independent experiments

Table 1 Identification of high molecular weight protein band increased in the parental cells

Protein Accession no Peptide count Unique
peptides

Confidence score M.W. (Dalton) Fold change
(treated/con- trol)

by label-free nLC-MS/MS

Heat shock 70 kDa protein 1A
Heat shock cognate 71 kDa protein

P0DMV8 25 18 200.8 70,337 15.5
P11142 22 17 168 71,126 1.16

The increased protein band was excised and analyzed by label-free nLC-MS/MS as detailed in the Material and Methods section. Peptide count: total identified peptides for the protein. Unique peptides: identified non-conflicting peptide sequences for the protein which the quantitation is based on. Confidence score: Identification score

solid tumors in near future. Heat shock proteins (HSPs) are expressed at high levels in a broad range of cancers and they are closely related to a poor prognosis and resistance to ther- apy [56, 57]. The results presented here showed that HSP70 is expressed more in the parental cells (a result observed even in coomassie blue-stained gels) than that observed in the resistant cells in response to varying bortezomib concen- trations. These results thus emphasize that the antiapoptotic HSP family members are not critical regulators of the bort- ezomib resistance; nonetheless, they are also responsive to the degree of the proteasomal inhibition as detected in the parental cells.

Altogether, it is concluded that ERK T202 phosphoryla- tion and autophagic activation are not the molecular phe- nomenon responsible for the resistance to bortezomib in our PC3 cell model. The results presented indicate that the ERK MAPK phosphorylation and autophagic activation are dependent on the degree of proteasomal inhibition. Finally, the combination of proteasome inhibitors with MELK inhib- itor OTSSP167 or with Ca2+ chelator BAPTA-AM may be a novel alternative treatment protocols for preventing prolif- eration of cancer cells with acquired or natural resistance to the proteasome inhibitors.

Fig. 7 Effect of MELK inhibitor OTSSP167, CDK2 inhibitor CVT-313, and BAPTA-AM
on the parental and resistant cells. The parental (PC3-P) and the resistant (PC3-R) cells were treated with 100 nM OTSSP167, 100 nM CVT-313,
and 20 µM BAPTA-AM for the indicated times after 24 of cell seeding. The proliferation status (cell index) was analyzed by
the iCELLigence system. The results are means ± SD (n = 2)
Acknowledgements The present study was financially supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK, Grant No. 119Z145).

Author contributions AY conceived and designed the experiments. AY, EK, and ATB carried out the experiments. AY and EK contributed to the interpretation and analysis of the results. AY and EK wrote the manuscript.

Data availability The data that support the findings of this study are available from the corresponding author (A.Y.) upon reasonable request.

Declarations

Conflict of interest The authors declare that they have no conflict of interest in relation to the study.

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