Marine alkaloid monanchoxymycalin C: a new specific activator of JNK1/2 kinase with anticancer properties

Monanchoxymycalin C (MomC) is a new marine pentacyclic guanidine alkaloid, recently isolated from marine sponge Monanchora pulchra by us. Here, anticancer activity and mechanism of action was investigated for the first time using a human prostate cancer (PCa) model. MomC was active in all PCa cell lines at low micromolar concentrations and induced an unusual caspase-independent, non-apoptotic cell death. Kinase activity screening identified activation of mitogen-activated protein kinase (MAPK) c-Jun N-terminal protein kinase (JNK1/2) to be one of the primary molecular mechanism of MomC anticancer activity. Functional assays demonstrated a specific and selective JNK1/2 activation prior to the induction of other cell death related processes. Inhibition of JNK1/2 by pretreatment with the JNK-inhibitor SP600125 antagonized cytotoxic activity of the marine compound. MomC caused an upregulation of cytotoxic ROS. However, in contrast to other ROS-inducing agents, co-treatment with PARP-inhibitor olaparib revealed antagonistic effects indicating an active PARP to be necessary for MomC activity. Interestingly, although no direct regulation of p38 and ERK1/2 were detected, active p38 kinase was required for MomC efficacy, while the inhibition of ERK1/2 increased its cytotoxicity. In conclusion, MomC shows promising activity against PCa, which is exerted via JNK1/2 activation and non-apoptotic cell death.

Flow cytometry analysis. The experiment was performed as previously reported 22 . 22Rv1 cells were seeded in 6-well plates (0.2 × 10 6 cells/well in 2 mL/well). Following overnight incubation the cells were pretreated with 100 µM z-VAD(OMe)-fmk for 1 h in 2 mL/well of fresh media. Censequantly, the cells were treated with the investigated drugs for 48 h and harvested by trypsinisation. Cells were immediately stained with annexin-V-FITC and propidium iodide and further analyzed using FACS Calibur instrument (BD Bioscience, San Jose, CA, USA). The results were quantified using the Cell Quest Pro software v. 5.2.1. (BD Bioscience).
Mtt assay. The experiment was performed as reported before 23 . 6,000 cells/well were seeded in a 96-well plate. Cells were incubated overnight and treated with MomC or vehicle (DMSO) in 100 μL/well of corresponding fresh culture media. Cells were consequently incubated with MTT solution (3-(-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 2-4 h. The culture media was removed and the plates were dried for 1 h. To dissolve the formazan crystals the 50 µL/well of 100% DMSO were added and the optical density was measured using a spectrophotometer Infinite F200PRO reader (TECAN, Männedorf, Switzerland).
Light microscopy. Cells were seeded in the 96-well plate and treated with the investigated drugs or vehicle for 48 h as described for MTT assay (see above). Microphotographs of the alive cells were taken using Axiovert 25 (Carl Zeiss, Göttingen, Germany) microscope, AxioCam MRc camera (Carl Zeiss) and AxioVision software v. 4.8.2 SP3 (Carl Zeiss) at 100 × magnification as previously reported 24 . The original images were croped and the figures were further prepared using the CorelDRAW X7 software v. 17.1.0.572 (Corel Corporation, Ottawa, Canada).
Colony formation assay. Colony formation assay was performed as previously reported 24 . 22Rv1 or PC-3 cells were seeded in ø 6 cm TC Petri dishes (Sarstedt, Numbrecht, Germany; 1 × 10 6 cells/dish in 5 mL/dish) and treated with indicated concentration of MomC for 48 h. Then, 100 alive cells were seeded in 6-well plates im 2 mL/well and incubated for 14 days. Surviving colonies were fixed with 100% MeOH, stained with Giemsa solution and counted by naked eye.
Western blotting. The experiment was performed as previously described 25 . In brief, 1 × 10 6 cells/dish cells were seeded in ø 6 cm Petri dishes in 5 mL/dish of media. Cells were incubated overnight followed by treatment with indicated concentrations of MomC or anisomycin for 48 h. Cells were harvested by scraping, washed (2 × ice cold PBS), and lysed. The samples were shortly frozen and centrifuged at 10,000g. The protein concentrations in the supernatants were determined by Bradford assay. The samples (20-30 μg/lane) were separated using SDS-PAGE method in gradient Mini-PROTEAN ® TGX Stain-FreeTM gels (Bio-Rad, Hercules, CA, USA) at 200 V. The proteins were transferred to a PVDF membrane, the membranes were blocked and consequantly incubated with the primary and secondary antibodies. The signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA). All procedures were performed according to the manufacturers' protocols. β-Actin was used as a loading control. For the list of used antibody see Supplementary Table S1. Kinase activity profiling. Kinase activity profiling was performed as described before 26 . For profiling serine-/threonine kinases, STK-PamChip ® arrays and a PamStation ® 12 (PamGene International, 's-Hertogenbosch, The Netherlands) were used according to the manufacturer's protocols. In brief, each array contains 140 individual peptide phospho-sites that are analogues of substrates for the corresponding serine-/threonine kinases. The whole cell lysates were prepared using M-PER Mammalian Extraction Buffer (Pierce, Waltham, Massachusetts, USA) containing Halt Phosphatase Inhibitor (Pierce) and EDTA-free Halt Protease Inhibitor Cocktail (Pierce). 1 µg of protein and 400 µM ATP were mixed and applied per each array. Sequence-specific peptide phosphorylation was detected using anti-phospho-Ser/Thr antibodies during the reaction followed by detection with secondary polyclonal swine anti-rabbit Immunoglobulin-FITC antibody (PamGene International). For the signal record a CCD camera and the Evolve software v. 1.0 (PamGene International). The quality of signals were controlled. For further data analysis the final signal intensities were log2-transformed and further proceeded using the BioNavigator software v. 6.0 (BN6, PamGene International).
Determination of drug combination effects. The effects (synergistic, antagonistic, or additive) of MAPK inhibitiors (PD98059, SCH772984, SP600125, SB203580), PARP inhibitior olaparib, androgen receptor antagonist enzalutamide, or antioxidant N-acetyl-l-cysteine (NaC) on the cytotoxic activity of MomC was deterimed using the Chou-Talalay method 27,28 . The experiments were performed as described before 9,25  Cell fractionation. The isolation of the cytosolic proteins was performed using the Cell Fractionation Kit (Cat. No ab109719, abcam, Cambridge, MA, USA) as reported before 22 . In brief, 22Rv1 cells were treated for 48 h and harvested by scratching. The further procedures were performed following the previously described protocol 29 . Generated cytosolic fraction was concentrated using Amicon ® Ultra-2 Centrifugal Filter device (Cat. . The values are presented as mean ± SEM. One-way ANOVA test followed by Sidak's multiple comparison test was used for group comparison. Differences were considered to be statistically significant: *p < 0.05; **p < 0.01; ***p < 0.001. All experiments were performed in triplicates.

Results
MomC is cytotoxic to human drug-resistant prostate cancer cells. The cytotoxic activity of MomC ( Fig. 1a) was investigated in the five prostate cancer cell lines PC-3, DU45, 22Rv1, VCaP, and LNCaP, possessing varying resistance profiles to standard therapies, as well as prostate non-cancer cell lines RWPE-1 and PNT2, and non-cancer human fibroblasts MRC-9 (Fig. 1b). Cytotoxicity was found in all cells tested at micro-or submicromolar concentrations following 48 h of treatment ( Fig. 1b). At the same time, no significant selectivity towards cancer cells was observed in the in vitro experiments (Fig. 1b). PC-3 and DU145 cells are known to be unresponsive to androgen receptor (AR)-targeting therapy due to a lack of AR expression 30 , while AR splice variant seven mediates resistance of 22Rv1 and VCaP cells due to a loss of the androgen binding site 30 . Of note, AR-V7 induces an activation of the AR pathway independently of androgen binding 31 . Thus, AR targeting drugs such as abiraterone and enzalutamide are mostly ineffective 31 . LNCaP is a hormone dependent cell line harboring the natural expression of the full length AR (AR-FL) 30  www.nature.com/scientificreports/ LNCaP cells by treatment with enzalutamide (AR antagonist) could antagonize the MomC cytotoxic activity (Fig. 1c). Therefore, we speculate that in prostate cancer cells MomC cytotoxicity correlates with AR activity. Colony formation of human prostate cancer cells was examined as a model for potential anti-metastatic activity of MomC. At non-cytotoxic concentrations the compound reduced the colony formation of 22Rv1 and PC-3 cells. Remarkably, active concentrations for PC-3 cells were 2-to 50-fold less than the cytotoxic IC 50 for the corresponding cell lines (Fig. 1d).

MomC induces non-apoptotic cell death of PCa cells. The mechanisms of MomC-induced cell death
were further investigated in drug-resistant 22Rv1 cells. 22Rv1 cells are known to be androgen-independent due to the expression of AR-V7, and therefore resistant to hormonal therapy 30 . Two apoptotic hallmarks, PARP cleavage and cleaved caspase-3, were detected after 48 h of MomC treatment (Fig. 2a), whereas anti-apoptotic protein survivin was downregulated (Fig. 2a). However, the morphology of the cells treated with MomC was markedly different from those treated with anisomycin (a well-established inducer of "classical" caspase-dependent apoptosis 32,33 or cisplatin. Thus, regular apoptotic features such as cell shrinking, membrane blebbing, and apoptotic bodies formation were not detected in MomC-treated cells (Fig. 2b); in contrast, cell rounding and swelling was observed under the treatment (Fig. 2b).
Furthermore, an increased annexin-V-FITC + /PI + and annexin-V-FITC − /PI + cell populations under the MomC treatment were observed by flow cytometry indicating fraction of late apoptotic and necrotic cells. Note, PI + staining indicates a loss of the cellular membrane integrity, being a feature of non-apoptotic cell death. At the same time, no significant increase of the Annexin-V-FITC + /PI − cell population (early apoptosis) was detected in the MomC-treated samples, while in contrast this cell fraction was observed secondary to anisomycin treatment (positive control) as expected (Fig. 2c). In order to determine the role of caspases in this process, a co-treatment with well-established pan-caspase inhibitor z-VAD(OMe)-fmk was applied. Interestingly, the inhibitor did not influence the cytotoxic effects of MomC, while the activity of anisomycin was significantly reduced. Expectedly, z-VAD(OMe)-fmk pretreatment resulted in an increase of the alive cell population and a decrease of the early and late apoptotic cell population of anisomycin treated cells (Fig. 2c). These results indicate that MomC-induced cell death was exerted via non-apoptotic mechanisms and independently from caspase activities.

MomC-induced alterations of protein tyrosine kinases activity.
Protein kinases catalyze the phosphorylation of specific proteins thereby modulating their activity. They play a critical role in numerous cancer-related events 34 . Unsurprisingly, they have become targets for anticancer therapeutics with many of them being clinically approved to date 34 . Since serine-/threonine kinases (STK) play a critical role in processes of cell death and survival, we systematically analyzed the STK activity performing functional kinomics 35 . We used the PamTechnology ® (https ://www.pamge ne.de, Fig. 3a) which allows the identification of kinases specifically activated or inhibited by the drug. For the experiment, a short drug exposure time of 2 h was chosen, to ensure, that only specific primary effects of MomC on the kinome of prostate cancer cells were identified, while the number of measured secondary effects resulting from cell death related processes was minimized. The results are presented as a log2 of signal intensity per peptide for each treatment group in Fig. 3b and summarized in Fig. 3c. The data indicate overall increased STK activity in MomC-treated samples compared to the control group (Fig. 3c, 3d). Note, no significant treatment-induced inhibition of phosphorylation was observed for any peptide tested (Fig. 3d).
Analyses of upstream kinases potentially influenced by MomC predicted the activation of several kinases belonging to the family of mitogen-activated protein kinase (MAPK), namely p38, JNK1/3, and ERK1/2-all known to be involved in different cancer-related processes 34,36 . In addition, a cardiac myosin light chain kinase (caMLCK), RSK-like pseudokinase 1 (RSKL1), and Y-linked protein kinase (PRKY) were predicted to be activated (Fig. 3e).
caMLCK is a kinase which is important for muscle contraction (in particular heart) 37 . There is few information on its role in cancer. Thus, a related kinase-i.e. non-muscular myosin light chain kinase (nmMLCK) was reported to be overexpressed in prostate cancer 38 . However, its role in cancer development and treatment seems to be controversial 39 . RSKL1 is a RSK-like pseudokinase 1, also known as ribosomal protein S6 kinase C1 (RPS6KC1). It may be involved in transmitting sphingosine-1 phosphate (SPP), which is an important cellular messenger 40 . Additionally, it can bind to peroxiredoxin-3 and may help to transport it to mitochondria and early endosomes 41 . However, no relation of this kinase with cancer progression or treatment prognosis was reported, even though the mutations of the correspondent gene were detected in several human cancers 42 . The activation of PRKY was predicted as well. The close analogue of this kinase, PRKX plays an important role in differentiation, epithelial morphogenesis, and is also involved in angiogenesis 43 . However, to the best of our knowledge, there is no known cellular function of PRKY as well as any reports on its expression in cancer cells.
Validation of kinome analysis data. All top-5 ranked kinases belong to the family of mitogen-activated protein kinases (Fig. 3e). Thus, p38, JNKs, and ERKs were predicted to be predominantly activated by MomC in cancer cells (Fig. 3e). In order to validate these findings, Western blotting analyses were performed for different MomC treatment periods (up to 48 h) (Fig. 4a). Indeed, a dose-and time-dependent activation of JNK1/2 was observed starting as early as 6 h after treatment initiation. In contrast, no caspase-3 activation was found at that time. Of note, no alterations of ERK1/2 or p38 activation were detected-even after 48 h of treatment. An inhibition of both phosphorylated and total p38 was attributed to cytotoxic processes, starting to be pronounced 12 h after treatment initiation (Fig. 4a).
Depending on the nature of the stimulus and the model used an activation of JNK1/2 and other MAPKs may exert either pro-apoptotic or pro-survival function 44 . In order to determine the specific effects in the prostate Scientific RepoRtS | (2020) 10:13178 | https://doi.org/10.1038/s41598-020-69751-z www.nature.com/scientificreports/ cancer cells, we performed co-treatment of the cells with an established JNK1/2 inhibitor SP600125. Due to the weak but still detectable cytotoxicity of SP600125 alone in 22Rv1 cells, a Chou-Talalay method was implemented.   www.nature.com/scientificreports/ index (CI). Hence, we created a heat-map of cytotoxic activity of MomC and SP600125 alone and in combination (Fig. 4b). Further analysis of the data revealed an antagonistic effect of SP600125 on MomC-induced cytotoxicity in 22Rv1 cells at the whole range of concentration of the investigated marine alkaloid (Fig. 4c). Thus, an activation of JNK1/2 under the treatment was determined to be a pro-cytotoxic stimulus.

Role of ROS and PARP in MomC-induced cells death.
As JNKs activation may result from oxidative stress 45 we examined ROS levels in MomC-treated 22Rv1 cells. Indeed, an upregulation of ROS was detected after 6 h of treatment (Fig. 5a). Moreover, pre-treatment of the cells with an established antioxidant N-acetyl-lcysteine (NaC) significantly antagonized cytotoxic effects of MomC (Fig. 5b). Thus, ROS activation significantly contributes to the anticancer activity of the investigated marine alkaloid. In line with this, we detected a release of cytotoxic mitochondrial proteins to cellular cytoplasm, e.g. cytochrome C and apoptosis inducing factor (AIF), which was correlating with the induction of cell death hallmarks (caspase-3-and PARP-cleavage) (Fig. 5c). This indicates MomC-induced mitochondria membrane permeabilization, which could be either a reason or a result of the elevated ROS production 46 . An excessive intracellular ROS may induce ssDNA break, which could, however, further be repaired by PARP, thereby rescuing a cancer cell from DNA damage-induced apoptosis 47 . Therefore, we examined the effect of PARP inhibitor olaparib on MomC activity. However, the expected synergistic effect of olaparib was not observed, but on the contrary, a pronounced antagonistic effect was detected (Fig. 5d). Of note, a similar effect was previously reported for ROS-induced non-apoptotic cell death, executed via consecutive induction of JNKs and sustained PARP1 activation 45 .

Role of other MAPK in MomC-induced cytotoxicity.
Next, we evaluated the impact of the pathways involving other MAPKs, predicted to be activated by kinome analysis (Fig. 3e), but without significant changes in the validation Western blotting analyses (Fig. 4a). Therefore, we applied p38 inhibitor SP203580 as well as ERK1/2 inhibitors FR180204 and SCH772984 in combination with MomC. Additionally, MEK1 inhibitor PD98039 was used. MEK1/2 kinase is known to directly and exclusively phosphorylate ERK1/2 48 . Thus, inhibition of MEK1/2 ultimately results in ERK1/2 inactivation 48 , and PD98039 is often used as an indirect ERK1/2 inhibitor. Due to the slight cytotoxic effects of these inhibitors alone, the Chou-Talalay method was used. We observed an antagonistic effect of SP203580 (p38i) on MomC activity in 22Rv1 cells (Fig. 6a). On the other hand, we detected synergistic effects of FR180204 (ERKi, Fig. 6b), SCH772984 (ERKi, Fig. 6c), as well as of PD98039  Fig. 6d) on the activity of the investigated alkaloid. This suggests that p38 kinase is involved in the cellular processes which are important for the MomC-induced cytotoxicity, whereas ERK1/2 mediates rather pro-survival processes (Fig. 6a-d).

Discussion
In the current study, we demonstrate cytotoxicity of recently isolated marine alkaloid MomC in PCa cells revealing different levels of resistance to approved standard therapies including taxanes and AR-targeting drugs such as abiraterone and enzalutamide. Effects were mediated by caspase-independent cytotoxic processes and exerted via activation of JNK1/2, a member of the mitogen-activated protein kinases (MAPKs). Protein kinases modify target proteins by phosphorylation resulting in changes of enzyme activity, cellular location and interaction with other proteins. Due to this key function they play an important role in cancer development and growth 49 . Among them, MAPKs are a prominent family well known to be involved in various cancer-related biological processes as well as development of drug resistance and depletion of oncogene-induced senescence 34 . For instance, c-Jun N-terminal protein kinases (JNKs)-a subset of MAPK-affect cell survival and death, proliferation, migration, differentiation and inflammation processes by interaction with different nuclear and non-nuclear proteins 50 . Typical JNKs' targets include c-Myc, p53, 14-3-3, Elk1, histone H3, histone H2AX, Bcl-2 family members, as well as several components of AP-1 (activator protein-1; a transcription factor) such as c-Jun and ATF2 (reviewed in Ref. 50 ). In cancer, JNKs have been described to be a "double-edged sword". Thus, in addition to a well-established and most often observed pro-apoptotic activity, anti-apoptotic functions were reported as well 44 . The effect of JNKs on cell viability seems to be dependent on the death stimulus' nature, cell type, duration of activation, and the status of the other signaling pathways 44 .
In prostate cancer, crucial effects on tumor growth have been assigned to specific members of the MAPK family 36,51,52 and tight interactions with AR-signaling have been demonstrated 53,54 -in particular, a crosstalk between AR and JNK 55 . Thus, activation of the AR pathway led to the suppression of JNK activation followed by inhibition of apoptosis in PCa cells 55 . In contrast, an active JNK was required to maintain the sensitivity of human PCa cells to androgens and androgen-receptor targeting drugs 56 . Moreover, anti-apoptotic activity of androgens in PCa were at least in part exerted via inhibition of JNKs 57 . Hence, JNK suppression contributes to androgen-mediated survival of PCa cells 57 . In addition, JNK inactivation or deletion promoted the development of aggressive androgen-independent metastatic prostate cancer in vivo. Thus, JNK inhibition seems to be an unfavorable event in prostate cancer therapy 58 . First clinical data confirm these findings. Thus, a higher intratumoral expression of active phospho-JNKs correlated with a survival advantage of prostate cancer patients 59 .
To date, both, intrinsic and extrinsic mediated apoptosis, as well as non-apoptotic/necrotic cell death have been reported following JNKs activation in PCa cells. While Bcl-2 phosphorylation and degradation, www.nature.com/scientificreports/ mitochondrial cytochrome C release and caspase-9 activation are involved in the intrinsic pathway 60 , extrinsic apoptosis is exerted by modulation of the TNFα-or FasL-Fas-mediated pathways 51,61 . In addition, endoplasmic reticulum (ER) targeting was described to result in JNKs activation resulting in apoptosis of prostate cancer cells (via Ca 2+ release induced JNK activation followed by the cleavage of executioner caspases; or ER stress induced IRE-1/ASK1/JNK signaling followed by intrinsic apoptosis) 51 . Non-apoptotic cell death was reported to be initiated under excessive ROS and executed via JNK-mediated PARP1 activation which ultimately lead to the cell death 45 .
In prostate cancer, approximately ten JNK-activating natural compounds (e.g. jungermannenone B 62 , capilliposide C 63 , guttiferone F 64 and others 51 ) have been identified to date and were reported to induce cell cycle arrest and apoptosis in vitro. Of note, for some of these substances, i.e. curcumin and costunolide, synergism with standard chemotherapeutics used in the treatment of prostate cancer was observed (reviewed in Ref. 51 ). In our study, the kinome screening identified MAPKs as potential direct or indirect targets of the marine natural compound MomC in PCa cells. Indeed, validation experiments revealed an activation of JNK1/2, which was relevant for MomC-induced cytotoxicity. Remarkably, this activation was observed shortly after treatment initiation and prior to cell death related events. Moreover, inhibition of JNKs activity by SP600125 effectively antagonized MomC cytotoxicity. Hence, JNK1/2 activation is not the result of the other cell death related events (e.g. caspases activation), but rather its initiator.
Remarkably, we previously reported the cytotoxic activity of MomC in human cervical carcinoma HeLa cells 21 . In that study an apoptosis-like character of the MomC-induced cell death was suggested 21 . Interestingly, in both, cervical 21 and prostate cancer cells (Fig. 2a-c) apoptotic hallmarks such as cleavage of caspase-3 and PARP were observed. However, more precise and detailed examinations suggested cancer cell elimination rather by non-apoptotic mechanisms, at least in PCa: this non-apoptotic character was indicated by (i) distinct morphology of the treated cells (cell rounding and swelling, lack of shrinking and blebbing, lack of apoptotic bodies formation), (ii) independence of the cytotoxic effect from caspase activity, (iii) increase of late apoptotic and necrotic cell fraction under MomC treatment, while no early apoptosis was detected. In fact, the detected caspase-3-and PARP-cleavage were most likely unspecific events secondary to other cell death related processes (e.g. mitochondria or lysosomal membrane permeabilization). Of note, the mechanism of the induced cytotoxic effects may be cell-specific and therefore distinct in the different models. The mode of MomC action in the human cervical carcinoma HeLa cells therefore awaits further examination and clarification.
Notably, we observed an upregulation of cytotoxic ROS and mitochondrial damage under drug exposure. In addition, active PARP was required for MomC-induced cytotoxic activity. Thus, the increased ROS production did not cause the classical apoptotic cell death mediated by the induction of ssDNA breaks, but rather a specific JNKs activation resulting in further PARP activation. Indeed, previous reports demonstrate that excessive ROS production leads to JNK activation followed by sustained PARP activation 45 . Consequences are a quick depletion of cellular NAD + resulting in ATP production failure and cell death of a non-apoptotic character 65 . Indeed, our findings indicate that MomC exerts its activity this way.
Interestingly, the cytotoxic activity of MomC in PCa correlated with the AR status of the cell lines, suggesting a functional AR pathway to contribute to the execution of the cytotoxic program. This can at least be partly explained by a cross-talk between AR and JNK. Indeed, JNK activation was reported to be downregulated with an increasing grade of PCa 57 . In addition, antiapoptotic activity of androgens in both androgen-dependent and -independent PCs was found to be executed via downstream blocking of JNK activation 55,57 . p38 and ERK1/2 are additional MAPK involved in different cellular processes, which mediate either procytotoxic or pro-survival processes depending on the stimuli and cellular context 36,49,66 . In prostate cancer, the activation of p38 by chemotherapy or other stress stimuli negatively regulates cell cycle progression, inhibits migration and promotes apoptosis (reviewed in Ref. 36 ). In contrast, in non-stress conditions p38 is critical for hypoxia-reoxygenation induced androgen-independent activation of AR, therefore contributing to the hormone resistance of PCa 67 . Moreover, an active p38 kinase contributes to survival, clonogenicity, migratory and invasive properties of PCa cells 36 . The role of ERK1/2 kinase in prostate cancer is described to be more univocal. Thus, it's basal activity as well as activation are associated with advanced malignancy, initiation of prostate cancer development and cancer cell invasion 36 . Moreover, ERK1/2 activation is associated with promotion of AR-signaling and increased PSA expression 36 . In patients, active ERK1/2 was found to be associated with advanced disease with the highest levels reported for metastatic castration-resistant PCa (mCRPC). First signs of clinical activity were reported for MEK/ERK inhibition and a phase II clinical trial examining the activity of trametinib in mCRPC is currently recruiting (NCT02881242) 68 .
In our experiments, the kinome analysis predicted p38 and ERK1/2 to be also regulated by MomC; however, no significant changes of the phosphorylated forms were observed in validation experiments. Of note, a distinct level of active p-ERK1/2 was detected in non-treated (control) cells, with no obvious further regulation under treatment. However, cellular processes controlled by ERK1/2 even at its basal activity level may counteract the activity of MomC. Thus, several ERK inhibitors synergized with MomC increasing its cytotoxic effects. Therefore, MEK/ERK inhibitors can be considered for a combinational use with MomC in future drug development. In contrast, an inhibition of p38 activity by p38 inhibitor SP203580 antagonized cytotoxic activity of MomC. At the same time, p38 was not activated under the MomC treatment. Hence, p38 activity (even without additional activation) may be important for successful execution of the MomC-induced cytotoxic program. Finally, it should be noted that a possible activation of caMLCK, predicted by kinome analysis, may cause a stimulation of heart muscle contraction. This potential side effect needs to be carefully monitored in in vivo experiments and future clinical trials.