- Email: [email protected]
Protein Kinase C regulates the complex between cell membrane molecules in ovarian cancer
Process Biochemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Protein Kinase C regulates the complex between cell membrane molecules in ovarian cancer Zehra Tavsana,b, Hulya Ayar Kayalia,c,d,* Izmir Biomedicine and Genome Center, 35340, İzmir, Turkey Department of Chemistry, The Graduate School of Natural and Applied Sciences, Dokuz Eylul University, 35390, İzmir, Turkey c Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, 35340, İzmir, Turkey d Department of Chemistry, Division of Biochemistry, Faculty of Science, Dokuz Eylul University, 35390, İzmir, Turkey a
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovarian cancer EpCAM Claudins Tetraspanins Protein kinase C
The interactions between the integrated complex array of integral and peripheral cell adhesion molecules (CAMs) are tightly controlled by kinases such as Protein Kinase C (PKC) in response to changes in external or internal forces and/or signaling. Focusing on the complex of EpCAM-claudin-tetraspanin-driven ovarian cancer, we described a sequence of events by which role of PKCs located in the tetraspanin enriched microdomains affected on the interactions and performed immunoprecipitations in PKC activator and inhibitors-treated ovarian cancer cells and xenograft ovarian cancer mouse models. Activated PKC isoforms associated with tetraspanins and induced detectable changes in the claudin phosphorylation state. These results suggest that PKC targets claudin-4 ad -7. Phosphorylation, especially by PKC δ and η of claudins was important for the interactions between claudin-4, -7 and EpCAM. These results represent the direct evidence that phosphorylation of claudins by PKCs functions in the EpCAM-claudin-tetraspanin complex formation to allow these complexes to operate in ovarian cancer progression and metastasis in vitro and in vivo.
1. Introduction During carcinogenesis, primary tumor cells lose their adhesion contacts, migrate through the lymphatic or blood system and form new tumors in the new metastasis sites. Therefore, tumor cells inevitably experience dysregulation in cell-cell adhesion, and the transformation of tumor cells is highly regulated by cell adhesion which occurs from the interactions between the different types of junctional complexes consisting of several cell adhesion molecules, cell adhesion molecules (CAMs) such as cadherins, integrins, cell surface proteoglycans, and tetraspanins on the cell surface. Type I and 39–42 kDa glycoprotein, Epithelial cell adhesion molecule (EpCAM) functions as a homophilic, epithelial-specific intercellular cell adhesion molecule [1–3] which is found expressed on a great variety of human carcinoma [4]. Also, EpCAM overexpression strongly correlates with worse overall survival, for instance in breast and ovarian cancer [5,6]. Among the most important structural and functional components of tight junctions, claudins are located in the mostly apical junctions of epithelial and endothelial cells and help to regulate paracellular permeability besides mediation to cell-to-cell adhesion. The aberrant expression of these proteins contributed to the destabilization of tight junctions and thus to
⁎
loss of cell polarity and cohesion [7–9]. In addition, tetraspanins can strongly influence cellular signaling such as adhesion, migration and invasion [10,11] by organizing laterally with other membrane proteins within the context of specialized tetraspanin-enriched microdomains (TEMs). Many studies have found correlations between tetraspanins and cancer progression [12–15]. The interactions between the integrated complex array of integral and peripheral CAMs which associate to several molecules involved in signaling cascades, as well as expressions are tightly controlled by kinases to facilitate junction assembly or disassembly. Phosphorylation acts as a switch on the cell adhesion molecules, turning “on” or “off” their interactions with other proteins. Phosphorylation which is catalyzed by kinases is a dynamic posttranslational modification and can alter protein structure, localization, protein-protein interactions and stability [16,17]. Protein Kinase C (PKC) family consists of at least 12 kinases and is involved in the phosphorylation of serine and threonine amino acid residues on the proteins. PKCs are translocated from cytoplasm to the membrane after activation by signals and play important roles in several signaling cascades ranging from cell adhesion, cellular growth, proliferation, differentiation and cell death [18,19]. Among gynecologic malignancies, ovarian cancer is the most lethal
Corresponding author at: Izmir Biomedicine and Genome Center, 35340, İzmir, Turkey. E-mail address: [email protected] (H.A. Kayali).
https://doi.org/10.1016/j.procbio.2020.01.009 Received 8 August 2019; Received in revised form 10 December 2019; Accepted 14 January 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Zehra Tavsan and Hulya Ayar Kayali, Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.01.009
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
incubated with specific primer antibodies of the studied molecules. After overnight incubation at 4 °C by rolling, the Protein A/G beads were added and incubated for 1 h at 4 °C. The immune complexes were collected by centrifuging at 14,000 rpm for 20 min 4 °C. The pellets were rinsed three times with lysis buffer. The cleared pellets were incubated in SDS-PAGE loading buffer at 95 °C for 20 min and maintained in the solubilized form by centrifuging.
as the majority will respond temporarily to surgery and cytotoxic agents and ranks fifth highest among cancer deaths for women. Due to complex and irrespective of the exact course of the pathogenesis of persistent and recurrent ovarian cancer, we previously initiated a serial analysis of the complex between cell adhesion molecules to better understand the molecular mechanisms of ovarian cancer. Co-immunoprecipitation analysis data revealed the EpCAM-claudin-4 or-7/ CD82-regulated ovarian cancer progression. From the significant body of our previous studies about the role of complex formation between EpCAM-claudin-4 or-7/CD82 in the ovarian cancer progression, better understanding of the role of this posttranslational modification, phosphorylation will provide important biologic and potentially therapeutic insights in the regulation of EpCAM-claudin-tetraspanin complexdriven ovarian cancer progression.
2.5. Western blotting After the last centrifugation step, the supernatants were mixed with SDS-PAGE loading buffer and equal amounts of total protein were loaded onto SDS-PAGE gels. SDS-PAGE was performed using a 10–12 % gel and the fractionated proteins were electroblotted onto nitrocellulose membranes. After blocking with 5 % BSA or skim milk powder in TBS with 0.1 % Tween 20 (TBST), membranes were incubated with against mono- or polyclonal antibodies of studied molecules. The membranes were washed in TBST and further incubated with HRP-conjugated secondary antibodies. The ECL Western blot analysis system was used to detect the antibodies. The density ratio of the specific bands was quantified by Image J program.
2. Materials and methods 2.1. Cell lines and cell culture The cell lines used were normal ovarian surface epithelial (OSE), A2780, OVCAR–3, SKOV–3 and A2780cis. Normal ovarian surface epithelial (OSE) cell line was purchased from Abm-Good. It was immortalized by SV–40 transfections. A2780, OVCAR–3, SKOV–3 and cisplatin-resistant A2780cis ovarian cancer cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC). The cells of passage 4 were resuscitated from liquid nitrogen stocks and cultured for less than 2 months before reinitiating culture from the same passage. ATCC, ECACC and Abm-Good had authenticated the cell lines using morphology, karyotyping, and PCR-based approaches.
2.6. Treatment of protein kinase C activator and inhibitors For studying the effects of PKCs on the interactions, 70–80 % confluent cells were incubated with 100 nm of PKC activator, PMA for 30 min at 37 °C. 5 μM of PKC δ inhibitor, rottlerin and PKC ƞ pseudosubstrate (PS) were treated for 2 h at 37 °C. 2.7. Xenograft animal studies
2.2. Cell growth conditions Six- to eight-week-old female BALB/c nude mice were provided by the Izmir Biomedicine and Genome Center (iBG) (Izmir, Turkey). The animals were housed in microisolator cages in a pathogen-free animal bio-safety level-2 facility at 22 ± 2 °C. Human SKOV-3 cells (5 × 106 cells/mice) were injected intraperitoneally (i.p.) into immunodeficient mice. Treatment started after 4 weeks of tumor formation. The mice were randomly divided into three groups (n = 6–8 per group). For the following two weeks, the mice were treated intraperitoneally with PBS, PKC ƞ PS- and rottlerin (2 times a week for 2 weeks). All procedures involving the use and care of mice were approved ethically and scientifically by the university in compliance with the Practice Guidelines for Laboratory Animals of Turkey. Formalin-fixed and paraffin-embedded tissues were sectioned (5μm-thick) and used to terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay for quantitative assessment of apoptosis. The sections were deparaffinated and dehydrated, and then TUNEL assay was carried out with an in situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol [20].
A2780, OVCAR–3 and A2780cis, SKOV–3 and OSE cell lines were grown in RPMI, McCoy’s 5A and Prigrow I growth mediums, respectively. All growth mediums contain 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin. The cells were incubated in CO2 incubator, conditioned to 37 °C and 5 % CO2 levels. For in vivo injection, SKOV-3 cells in logarithmic growth phase were trypsinized and centrifuged at 800 g for 5 min, washed twice, reconstituted in PBS at a concentration of 5 × 106 cells/mice for intraperitoneal injections. 2.3. The preparation of cell and tissue lysates The cells, at confluence about 80–90 % were washed with cold phosphate buffered saline PBS. The cells were collected by cell scraper and pelleted after centrifuging at 2000 rpm for 5 min. The pellets were lysed in the lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl) including the appropriate detergent (1 % Triton X-100, Brij 96 and CHAPS) and supplemented with aprotinin, leupeptin and phenyl-methyl-sulfonylfluoride (PMSF) as proteases and phosphatases inhibitors. After sonication, the lysates were incubated in the lysis buffer on ice for 15 min and centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatants were used for western blotting and immunoprecipitation. The protein levels were determined with BCA assay. The absorbance was read at 562 nm. BSA standard was used for calibration. Total proteins were purified from snap frozen tumors after homogenization in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 % Triton X-100 in the presence of protease and phosphatase inhibitors. Lysates were cleared by centrifuging at 13,300 rpm for 20 min and protein concentration was determined using a BCA kit and continued with immunoprecipitation and western blotting.
2.8. Statistical analysis SPSS20.0 software was used to input and analyze data, measurement data comparison between two groups was by t-test and P < 0.05 indicated statistical significance in differences. 3. Results We previously initiated a serial analysis of the complex between cell adhesion molecules to better understand the molecular mechanisms of ovarian cancer using different cell lines which have isolated primary and metastatic solid tumors, and ascites fluid. Co-immunoprecipitation analysis data revealed the EpCAM-claudin-4 or-7/CD82-regulated ovarian cancer progression. At the first step of the study, we evaluated which PKC isoforms were located with tetraspanin, CD82 which formed complexes with EpCAM and claudin-4 or -7. Then, how the
2.4. Immunoprecipitation The preparation of the supernatants mentioned in the previous section. The supernatants containing about 750 μg protein were 2
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
Fig. 1. Phosphorylation modulates the interactions. (A) A2780, OVCAR-3 and SKOV-3 cells were treated with PMA and then the proteins were immunoprecipitated with the indicated antibodies. Then, the interacted PKC isoforms was confirmed by immunoblot. After the treatments with PKC η pseudosubstrate (PS) (B) and rottlerin (C) in PMA-induced A2780, OVCAR-3 and SKOV-3 cells, lysates were immunoprecipitated with the indicated antibodies. The interactions were confirmed by immunoblot.
and PKC δ in OVCAR-3 and SKOV-3 cells (Fig. 1A). In A2780 cells, the interactions between CD151 and PKC η were not determined. Further studies continued with PKC η and PKC δ among the studied PKC isoforms because the no coimmunoprecipitation with PKC α and β1. In order to investigate whether PKC changes EpCAM, claudins and tetraspanins interactions, we treated ovarian cancer cells with the PKC activator, PMA and PKC inhibitors, and examined claudin-4 and -7, EpCAM and CD82 status in claudin-4 and -7 immunoprecipitants. Fig. 1B and C showed the interactions of claudin-4 and claudin-7 with claudin-4, -7, EpCAM and CD82 after PKC η PS and rottlerin treatment compared with PMA treatment. PKC η pseudosubstrate (PS) and rottlerin specifically inhibit PKC η and δ, respectively. Without activated PKCs, immunoprecipitants of claudin-7 from A2780 cells yielded no associated claudin-4. When compared with PMA treatment, the amounts of claudin-7, -4 and CD82 were significantly decreased in claudin-4 immunoprecipitants of OVCAR-3 and SKOV-3 cells after PKC η PS treatment. Similarly, the interactions between claudin-7, -4 and CD82 with claudin-7 were diminished with PKC η inhibition. Unexpectedly, claudin-4 or claudin-7 interactions with EpCAM did not change after PKC η PS treatment. When compared with PMA treatment,
phosphorylation by PKCs affects EpCAM-claudin-4 or-7/CD82 complex was investigated by using PKC inhibitors. 3.1. The interactions of claudin are regulated by PKCs When considering regulatory mechanisms which determine assembly, remodeling/modulation, and dynamics of cell adhesion, it occurs on various levels. Interestingly, several studies demonstrated the involvement of various kinases in the phosphorylation and regulation of proteins. The phosphorylation determined the activity and interactions of a protein at the plasma membrane where numerous molecules of cell signaling pathways interact within. Starting from the consequences that PKCs associate closely with several different tetraspanin proteins upon activation and translocation, we first evaluated which PKC isoforms were located with associated tetraspanins by western blotting of PKC α, β1, δ, and η in CD82 and CD151 immunoprecipitants after activation of PKC isoforms with PMA. In the CD82 and CD151 coimmunoprecipitates of A2780, OVCAR-3 and SKOV-3 cells, PKC α and β1 were not observed (data not shown because of no bands). The CD82 and CD151 coimmunoprecipitated with PKC η 3
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
-7 by serine and threonine residues which are phosphorylated by serine and threonine kinase, PKCs compared to the cells treated with PMA and rottlerin (Fig. 3).
the amounts of claudin-4, -7 and EpCAM in the claudin-4 and claudin-7 immunoprecipitants decreased after rottlerin treatment in A2780 cells. In OVCAR-3 cells treated with rottlerin, interactions between claudin-7 and claudin-4 and, also the amounts of EpCAM in the claudin-7 immunoprecipitants were completely lost, suggesting that phosphorylated claudin-7 is the most important for the complex formation between claudin-4, -7 and EpCAM. In addition, after PKC δ inhibition with rottlerin, the claudin-4 and EpCAM amounts in claudin-4 immunoprecipitants were significantly decreased. In contrast, PKC δ inhibition decreased the amounts of claudin-4, -7 and CD82 in claudin-4 and -7 immunoprecipitants. When all results were summed, phosphorylation, especially by PKC δ and η was important for the interactions between claudin-4, -7 and EpCAM in the metastatic ovarian cell lines, OVCAR-3 and SKOV-3. Unfortunately, the ovarian cancer was diagnosed at the late stage. In addition, in accordance to the main point which is to study PKC regulation of ovarian cancer driven by EpCAMtetraspanin-claudin complex and also in the light of the recent studies of PKC δ as a prime candidate [21–23], the further studies continued with PKC δ in OVCAR-3 and SKOV-3 cells.
3.3. In vivo EpCAM-claudin-tetraspanin complex formation EpCAM, claudin-4 and -7, and CD82 coexpression correlated with the metastasis and drug resistance in ovarian cancer. To reassure the in vivo relevance of complex formation, coimmunoprecipitation of EpCAM, claudin-4 and -7, and CD82 were evaluated in the harvested tumor tissues of xenograft ovarian cancer mice. Given the absence of certain cell number on xenograft ovarian cancer models in the literature, it has been difficult to determine the cancer cell number. In our preliminary studies, we evaluated the distribution of a various number of intraperitoneal (i.p.) injected ovarian cancer cells during 4 weeks. The results in 2 × 106 cell-injected nude mice exhibited the quite small and slow-growing tumors, usually around the injection site without losing weight, proving any antisurvival effect on the nude mice. The tumors in 5 × 106 cell-injected nude mice were metastasized on the liver surface, diaphragm and colon through the peritoneal cavity. In contrast, the tumors were too large to adversely affect the survival of the animals in 10 × 106 cells-injected mice. We chose the 5 × 106 to assess the complex formation in in vivo. In line with the in vitro findings, we noted in vivo experiments that EpCAM, claudin-4 and -7, and CD82 formed a complex in TEM (Fig. 4A). Analyzing the coexpression of pairs of these molecules significantly strengthened the difference between metastatic tumors and primary tumors (data not shown). Fig. 4B showed that PKC η and δ coimmunoprecipitated with CD82 and CD151, respectively, proving the direct interaction of activated PKC with tetraspanins in TEMs. In the tumors of PKC ƞ PS- and rottlerin-treated groups, EpCAM, claudin-4 and -7, and CD82 interactions were abrogated by PKC inhibition compared to the control PMA-treated mice (Fig. 4C). To confirm the hypothesis that progression of ovarian cancer could
3.2. Claudin-4 and -7 specifically are phosphorylated by PKC δ The experiments compared to PKC activator and inhibitors treatments suggested PKC δ as a prime candidate for the phosphorylation of EpCAM and claudins, which molecules were known phosphorylated by PKCs [21,22,24]. In order to verify whether claudin-4 and -7 could be phosphorylated by PKC, ovarian cancer cells (OVCAR-3 and SKOV-3) were treated with the PKC activator and inhibitor, and phosphorylation status were examined. PKC activation induced detectable changes in the claudin phosphorylation state suggests that PKC targets claudin-4 ad -7 (Fig. 2). When claudin-4 and -7 immunoprecipitations PMA- and rottlerintreated cancer cells compared, it revealed that PKC inhibition significantly downregulated the level of phosphorylation in claudin-4 and
Fig. 2. Claudins-interacted with EpCAM and tetraspanins are phosphorylated by PKCs. PMA-activated and rottlerin-inhibited OVCAR-3 (A), SKOV-3 (B) and A2780cis (C) cells were immunoprecipitated with claudin-4 and -7 and immunoblotted for phosphothreonine and phosphoserine. 4
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
Fig. 3. Relative band intensity of phosphoserine and phosphothreonine bands of claudin-4 and -7 immunoprecipitants in PMA- and rottlerin-treated OVCAR-3 and SKOV-3 cells.
Protein Kinase C (PKC), a family of phospholipid-dependent serine/ threonine kinases plays key roles in many of the signaling pathways [25,26]. In parallel with the activation of cytosolic PKCs, they translocate to the cellular membranes. Various PKCs also associate with the specific binding proteins in the membrane [27]. However, aside from PKC interaction with the transmembrane proteoglycan syndecan-4 [28], a role for specific transmembrane proteins such as tetraspanins during PKC translocation has previously been suggested. Termini et al. showed that the translocation of PKCs to the plasma membrane leads to their specific association with tetraspanin proteins [29]. Similarly, our findings indicated that CD82 which recruited EpCAM and claudins in the TEMs was also associated with specific PKCs under PKC activation. The possible location of tetraspanin-PKC complexes in TEMs, coupled with the appearance of complexes only under mild detergent conditions, suggests weak interactions. In addition, due to translocation of PKCs to the plasma membrane in the TEMs via activation, the molecules which already constitutively associated with tetraspanin proteins become linked to PKC and these associated molecules are phosphorylated in a PKC-dependent manner. Our experiments under PKC-activated and -inhibited conditions revealed that PKC phosphorylation leads to the formation of EpCAM-claudin-tetraspanin complex. Zhang et al. indicated although formation and maintenance of tetraspanins-PKC complexes are not dependent on integrins, tetraspanins proteins can act as linker molecules, recruiting PKC into proximity with specific integrins [30]. Also, claudin-7 associated with EpCAM, CD44v6 and D6.1A and the functional tumor progression activity of EpCAM-claudin7 complex in pancreatic and colorectal carcinomas depends on claudin7 phosphorylation [31]. In the current work, we have examined the claudin-4 and -7 phosphorylation by PKC, as consequences of abrogated interactions between EpCAM, claudins and tetraspanins in PKC inhibitors-treated ovarian cancer cells. We showed that the inhibition patterns of claudin-4 and -7 phosphorylation in the presence of the PKC δ inhibitor, rottlerin which the results were consistent with the studies of D'Souza et al., indicating that phosphorylation of claudin-3 and claudin-4 by PKA and PKC ε, respectively in ovarian cancer cells [22,32]. Thus, the results provide the first evidence for the phosphorylation of endogenous claudin-4 and -7 by PKC δ in the ovarian cancer cells, and depending on the phosphorylation status, EpCAM-claudinstetraspanins complex was observed or not. Besides, the current findings were consistent with the studies that have previously shown that posttranslation modifications by phosphorylation of the overexpressed claudins may inhibit their tight junction-related function while allowing other pro-oncogenic effects. Because claudins in the tight junctions found in the lipid rafts as characterized detergent-insoluble
be stopped if the complex formation was impaired by PKC inhibition, the growth-inhibitory effects of PKC inhibitors on ovarian cancer were examined in vivo. For this purpose, i.p. xenograft ovarian cancer models were employed and the effects of PKC inhibitors on the intra-abdominal dissemination of ovarian cancer tumors, ascites formation, and tumor growth were examined. Drugs showed no apparent toxicity throughout the study. The mice treated with PMA had significant abdominal swelling because of ascites formation, whereas swelling was much less in mice treated with PBS and PKC inhibitors (data not shown). At 2 weeks after initiation of treatment, postmortem examination showed that the tumors were on the surface of the peritoneum, intestines, omentum, and uterus in the PKC inhibitors treated groups. However, in the control PMA treated groups, in addition to the peritoneum, intestines, omentum, and uterus-disseminated tumors, the tumors on the diaphragm and/or the hilus of the liver varied. Especially, the tumor distribution in rottlerin-treated mice significantly decreased compared both PMA- and PKC ƞ PS-treated groups (data not shown). In spite of the reduction of metastasis to the distant organs, tumor volumes increased significantly in the PKC inhibitor-treated group compared to PMA-treated group (Fig. 4D). Because, there was the inflammation in the tumors of PKC inhibitor-treated groups (data not shown). To gain insight into the mechanism of rottlerin-dependent tumor metastasis reduction in vivo, apoptosis was assessed in the harvested tumors from rottlerin- and PKC ƞ PS-treated mice compared to the PMA and PBS-treated groups by TUNEL analysis. As shown in Fig. 4E, there were many TUNEL-positive cells inside of the small tumors obtained on the liver and diaphragm while apoptosis started from the outer side of tumors tissue taken from rottlerin-treated mice. In addition, in PKC ƞ PS-treated mice groups, TUNEL positive cells were observed. TUNEL staining was negative in PBS- and PMA-treated groups. These results indicated that PKC inhibition induced apoptosis. However, the relation between apoptosis, proliferation and metastasis of PKC inhibitors have to be further studied. 4. Discussion Epithelial cells adhere to their neighbors and the extracellular matrix, mediated by different types of junctional complexes. These complexes are tightly controlled by kinases to facilitate junction assembly or disassembly. Phosphorylation acts as a switch on the cell adhesion molecules, turning “on” or “off” their interactions with other proteins by altering protein structure, localization, protein-protein interactions and stability. Recent studies highlight the regulation of cell adhesiondriven signaling during cancer progression and metastasis. 5
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
Fig. 4. The interactions in tumors of xenograft ovarian cancer mice. After 4 weeks, the tumours were taken from the animals and total proteins were purified from snap frozen tumors by homogenisation in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % Triton X-100. The indicated molecules were immunoprecipitated and analysed by western blotting (A). The lysates of control mice were immunoprecipitated with the indicated antibodies. Then, the interacted PKC isoforms was confirmed by immunoblotting (B). The xenograft ovarian cancer mice were treated with PMA and PKC inhibitors for 2 weeks and the lysates of snap frozen tissues were obtained as indicated in Material and method section. The proteins were immunoprecipitated with the indicated antibodies. Then, the interacted molecules were confirmed by immunoblotting (C). Appearance of tumors after treatment of mice with PKC ƞ PS and rottlerin. Four representative mice inoculated with SKOV-3 cells treated i.p. with PBS (control), PKC ƞ PS and rottlerin. Treatments were started 2 weeks after inoculation with SKOV-3 cells. At the end of the experiment (2 weeks of treatment), mice were sacrificed. At autopsy, tumors were excised and ascites fluid was collected (D). Rottlerin- and PKC η pseudosubstrate (PS) induced cell apoptosis in tumor tissues in vivo was measured by TUNEL assay compared with PMA- and PBS-treated xenograft ovarian cancer mice. The representative images are shown with TUNEL signals (green) (E) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
activity. Only when complexes are located in glycolipid-enriched membrane domains, cells have displayed increased apoptosis resistance and an increased tendency to aggregate [34]. Cell culture experiments with ovarian cancer cells which have
fraction [33], to display the pro-oncogenic functions, overexpression, complex formation with EpCAM and tetraspanins of claudins, and also complex location in the detergent-soluble glycolipid-enriched membrane domains outside of tight junctions is crucial for its functional 6
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
different character showed that coexpression of EpCAM, claudins and tetraspanin in the glycolipid-enriched microdomains, TEMs was related to ovarian cancer progression and also cisplatin resistance. Thus, we asked whether the coexpression of these markers might provide a diagnostic and/or prognostic variable. In line with our hypothesis that to mimic metastatic stage in ovarian cancer patients, we generated xenograft ovarian cancer model by intraperitoneal injection of metastatic SKOV-3 cells. The immunoprecipitation experiments of xenograft tumors demonstrated similar results with cell line experiments. Similarly, in the experiments of Schmidt et al. and Ladwein et al., the metastasizing gastrointestinal tumors of the rat frequently express a complex that includes the tetraspanin D6.1A, EpCAM, claudin-7, and CD44v6 [31,34]. The evaluation of colorectal cancer and liver metastasis patients, as well as tumor-free colon and liver tissue showed that coexpression and complex formation of the EpCAM, claudin-7, CO-029, and CD44v6 molecules was correlated with clinical variables and apoptosis resistance [35]. The involvement of EpCAM, CD44v6 and claudin-7 in the progression of thyroid cancer from an indolent to an aggressive phenotype has been showed [36]. In accordance with our findings, PKC inhibitor, chelerythrine treatment of nude mice bearing HNSCC resulted in significant tumor growth delay [37]. Also, combined inhibition of PKC and histone deacetylase 1 lowered drug doses needed in vitro, in vivo, and ex vivo in patient-derived advanced basal cell carcinomas explants [38]. In whole lung tumors, fluorescence microscopy quantified double-positive green/red tumor cells experiencing caspase cleavage after treatment with PKC inhibitors, proving that PKC activation was significantly linked to the survival of highly metastatic cells [39]. Spleen and liver tissue sections of BALB/c mice treated with thymoquinone showed an increased number of apoptotic cells [40]. Collectively, we showed a novel mechanism which is responsible for ovarian cancer progression in vitro and in vivo. Our findings assigned PKC-modulated EpCAM-claudin-4/-7-CD82 complex. The disruption of these complex as a result of PKC inhibition or different approaches is a good candidate to stop or slow down ovarian cancer invasion, metastasis and drug resistance.
[3]
[4]
[5]
[6]
[7] [8] [9]
[10] [11] [12] [13]
[14]
[15]
[16]
[17]
[18]
Funding
[19]
This work was supported by TUBITAK (The Scientific and Technological Research Council of Turkey-113Z641 project number).
[20]
Author statement [21]
Conception and design: H. Ayar Kayali Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z. Tavsan Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Tavsan Writing, review, and/or revision of the manuscript: H. Ayar Kayali and Z. Tavsan
[22]
[23]
[24]
Declaration of Competing Interest
[25]
No potential conflicts of interest were disclosed.
[26]
Acknowledgements [27]
We gratefully thank the Graduate School of Natural and Applied Sciences, and İzmir Biomedicine and Genome Center.
[28]
References
[29]
[1] P.A. Baeuerle, O. Gires, EpCAM (CD326) finding its role in cancer, Br. J. Cancer 96 (2007) 417–423, https://doi.org/10.1038/sj.bjc.6603494. [2] M. Balzar, I.H. Briaire-de Bruijn, H.A.M. Rees-Bakker, F.A. Prins, W. Helfrich, L. de Leij, G. Riethmuller, S. Alberti, S.O. Warnaar, G.J. Fleuren, S.V. Litvinov, Epidermal
[30]
[31]
7
growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions, Mol. Cell. Biol. 21 (2001) 2570–2580, https:// doi.org/10.1128/MCB.21.7.2570-2580.2001. S.V. Litvinov, M.P. Velders, H.A.M. Bakker, G.J. Fleuren, S.O. Warnaar, Ep-CAM: A human epithelial antigen is a homophilic cell-cell adhesion molecule, J. Cell Biol. 125 (1994) 437–446, https://doi.org/10.1083/jcb.125.2.437. L. Huang, Y. Yang, F. Yang, S. Liu, Z. Zhu, Z. Lei, J. Guo, Functions of EpCAM in physiological processes and diseases (Review), Int. J. Mol. Med. 42 (2018) 1771–1785, https://doi.org/10.3892/ijmm.2018.3764. T. Hiraga, S. Ito, H. Nakamura, EpCAM expression in breast cancer cells is associated with enhanced bone metastasis formation, Int. J. Cancer 138 (2016) 1698–1708, https://doi.org/10.1002/ijc.29921. J. Zheng, L. Zhao, Y. Wang, S. Zhao, M. Cui, Clinicopathology of EpCAM and EGFR in human epithelial ovarian carcinoma, Open Med. 12 (2017) 39–44, https://doi. org/10.1515/med-2017-0007. S. Tabariès, P.M. Siegel, The role of claudins in cancer metastasis, Oncogene 36 (2017) 1176–1190, https://doi.org/10.1038/onc.2016.289. M.J. Kwon, Emerging roles of claudins in human cancer, Int. J. Mol. Sci. 14 (2013) 18148–18180, https://doi.org/10.3390/ijms140918148. S. Tabariès, A. McNulty, V. Ouellet, M.G. Annis, M. Dessureault, M. Vinette, Y. Hachem, B. Lavoie, A. Omeroglu, H.G. Simon, L.A. Walsh, S. Kimbung, I. Hedenfalk, P.M. Siegel, Afadin cooperates with claudin-2 to promote breast cancer metastasis, Genes Dev. 33 (2019) 180–193, https://doi.org/10.1101/gad. 319194.118. C.M. Termini, J.M. Gillette, Tetraspanins function as regulators of cellular signaling, Front. Cell Dev. Biol. 5 (2017), https://doi.org/10.3389/fcell.2017.00034. X. Jiang, J. Zhang, Y. Huang, Tetraspanins in cell migration, Cell Adhes. Migr. 9 (2015) 406–415, https://doi.org/10.1080/19336918.2015.1005465. M.E. Hemler, Tetraspanin proteins promote multiple cancer stages, Nat. Rev. Cancer 14 (2014) 49–60, https://doi.org/10.1038/nrc3640. F.Q. Hou, X.F. Lei, J.L. Yao, Y.J. Wang, W. Zhang, Tetraspanin 1 is involved in survival, proliferation and carcinogenesis of pancreatic cancer, Oncol. Rep. 34 (2015) 3068–3076, https://doi.org/10.3892/or.2015.4272. J. Lu, J. Li, S. Liu, T. Wang, A. Ianni, E. Bober, T. Braun, R. Xiang, S. Yue, Exosomal tetraspanins mediate cancer metastasis by altering host microenvironment, Oncotarget 8 (2017) 62803–62815, https://doi.org/10.18632/oncotarget.19119. R.R. Malla, S. Pandrangi, S. Kumari, M.M. Gavara, A.K. Badana, Exosomal tetraspanins as regulators of cancer progression and metastasis and novel diagnostic markers, Asia J. Clin. Oncol. 14 (2018) 383–391, https://doi.org/10.1111/ajco. 12869. K. Aoki, K. Yoshida, Biological consequences of priming phosphorylation in cancer development, Protein Phosphorylation (2017), https://doi.org/10.5772/ intechopen.70039. F. Ardito, M. Giuliani, D. Perrone, G. Troiano, L. Lo Muzio, The crucial role of protein phosphorylation in cell signalingand its use as targeted therapy (Review), Int. J. Mol. Med. 40 (2017) 271–280, https://doi.org/10.3892/ijmm.2017.3036. A.C. Newton, Protein kinase C: perfectly balanced, Crit. Rev. Biochem. Mol. Biol. 53 (2018) 208–230, https://doi.org/10.1080/10409238.2018.1442408. A. Tarafdar, A.M. Michie, Protein kinase C in cellular transformation: A valid target for therapy? Biochem. Soc. Trans. 42 (2014) 1556–1562, https://doi.org/10.1042/ BST20140255. C. Pu, S. Chang, J. Sun, S. Zhu, H. Liu, Y. Zhu, Z. Wang, R.X. Xu, Ultrasoundmediated destruction of LHRHa-targeted and paclitaxel-loaded lipid microbubbles for the treatment of intraperitoneal ovarian cancer xenografts, Mol. Pharm. 11 (2014) 49–58, https://doi.org/10.1021/mp400523h. N. Maghzal, H.A. Kayali, N. Rohani, A.V. Kajava, F. Fagotto, EpCAM controls actomyosin contractility and cell adhesion by direct inhibition of PKC, Dev. Cell 27 (2013) 263–277, https://doi.org/10.1016/j.devcel.2013.10.003. T. D’Souza, F.E. Indig, P.J. Morin, Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells, Exp. Cell Res. 313 (2007) 3364–3375, https://doi.org/10.1016/j.yexcr.2007.06.026. S. Jaken, P.J. Parker, Protein kinase C binding partners, Bioessays 22 (2000) 245–254, https://doi.org/10.1002/(SICI)1521-1878(200003)22:3<245::AIDBIES6>3.0.CO;2-X. C.M. Van Itallie, J.M. Anderson, Phosphorylation of tight junction transmembrane proteins: many sites, much to do, Tissue Barriers 6 (2018), https://doi.org/10. 1080/21688370.2017.1382671. G. Chandrika, K. Natesh, D. Ranade, A. Chugh, P. Shastry, Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling, Sci. Rep. 6 (2016), https://doi.org/10.1038/srep22455. S.M.A. Islam, R. Patel, M. Acevedo-Duncan, Protein Kinase C-ζ stimulates colorectal cancer cell carcinogenesis via PKC-ζ/Rac1/Pak1/β-Catenin signaling cascade, Biochim. Biophys. Acta - Mol. Cell Res. 1865 (2018) 650–664, https://doi.org/10. 1016/j.bbamcr.2018.02.002. S.J. Van Deventer, V.M.E. Dunlock, A.B. Van Spriel, Molecular interactions shaping the tetraspanin web, Biochem. Soc. Trans. 45 (2017) 741–750, https://doi.org/10. 1042/BST20160284. E.S. Oh, A. Woods, J.R. Couchman, Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C, J. Biol. Chem. 272 (1997) 8133–8136. C.M. Termini, K.A. Lidke, J.M. Gillette, Tetraspanin CD82 Regulates the spatiotemporal dynamics of PKCα in acute myeloid leukemia, Sci. Rep. 6 (2016), https:// doi.org/10.1038/srep29859. S. Hwang, T. Takimoto, M.E. Hemler, Integrin-independent support of cancer drug resistance by tetraspanin CD151, Cell. Mol. Life Sci. 76 (2019) 1595–1604, https:// doi.org/10.1007/s00018-019-03014-7. D.S. Schmidt, P. Klingbeil, M. Schnölzer, M. Zöller, CD44 variant isoforms associate
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
[32]
[33]
[34]
[35]
[36]
with tetraspanins and EpCAM, Exp. Cell Res. 297 (2004) 329–347, https://doi.org/ 10.1016/j.yexcr.2004.02.023. T. D’Souza, R. Agarwal, P.J. Morin, Phosphorylation of Claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells, J. Biol. Chem. 280 (2005) 26233–26240, https://doi.org/10. 1074/jbc.M502003200. A. Nusrat, C.A. Parkos, P. Verkade, C.S. Foley, T.W. Liang, W. Innis-Whitehouse, K.K. Eastburn, J.L. Madara, Tight junctions are membrane microdomains, J. Cell. Sci. 113 (2000) 1771–1781. M. Ladwein, U.F. Pape, D.S. Schmidt, M. Schnölzer, S. Fiedler, L. Langbein, W.W. Franke, G. Moldenhauer, M. Zöller, The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7, Exp. Cell Res. 309 (2005) 345–357, https://doi.org/10.1016/j.yexcr.2005.06.013. S. Kuhn, M. Koch, T. Nübel, M. Ladwein, D. Antolovic, P. Klingbeil, D. Hildebrand, G. Moldenhauer, L. Langbein, W.W. Franke, J. Weitz, M. Zöller, A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression, Mol. Cancer Res. 5 (2007) 553–567, https://doi.org/10.1158/ 1541-7786.MCR-06-0384. T. Okada, T. Nakamura, T. Watanabe, N. Onoda, A. Ashida, R. Okuyama, K.I. Ito, Coexpression of EpCAM, CD44 variant isoforms and claudin-7 in anaplastic thyroid
[37]
[38]
[39]
[40]
8
carcinoma, PLoS One 9 (2014) 1–13, https://doi.org/10.1371/journal.pone. 0094487. S.J. Chmura, M.E. Dolan, a Cha, H.J. Mauceri, D.W. Kufe, R.R. Weichselbaum, In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo, Clin. Cancer Res. 6 (2000) 737–742. A.N. Mirza, M.A. Fry, N.M. Urman, S.X. Atwood, J. Roffey, G.R. Ott, B. Chen, A. Lee, A.S. Brown, S.Z. Aasi, T. Hollmig, M.A. Ator, B.D. Dorsey, B.R. Ruggeri, C.A. Zificsak, M. Sirota, J.Y. Tang, A. Butte, E. Epstein, K.Y. Sarin, A.E. Oro, Combined inhibition of atypical PKC and histone deacetylase 1 is cooperative in basal cell carcinoma treatment, JCI Insight 2 (2017), https://doi.org/10.1172/jci. insight.97071. S.-H. Hong, L. Ren, A. Mendoza, A. Eleswarapu, C. Khanna, Apoptosis resistance and PKC signaling: distinguishing features of high and low metastatic cells, Neoplasia 14 (2012) 249–258, https://doi.org/10.1593/neo.111498. L.Z. Ali Salim, R. Othman, M.A. Abdulla, K. Al-Jashamy, H.M. Ali, P. Hassandarvish, F. Dehghan, M.Y. Ibrahim, F.A.E. Ahmed Omer, S. Mohan, Z. Wang, Thymoquinone inhibits murine leukemia WEHI-3 Cells in vivo and in vitro, PLoS One 9 (2014) e115340, , https://doi.org/10.1371/journal.pone.0115340.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Protein Kinase C regulates the complex between cell membrane molecules in ovarian cancer Zehra Tavsana,b, Hulya Ayar Kayalia,c,d,* Izmir Biomedicine and Genome Center, 35340, İzmir, Turkey Department of Chemistry, The Graduate School of Natural and Applied Sciences, Dokuz Eylul University, 35390, İzmir, Turkey c Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, 35340, İzmir, Turkey d Department of Chemistry, Division of Biochemistry, Faculty of Science, Dokuz Eylul University, 35390, İzmir, Turkey a
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ovarian cancer EpCAM Claudins Tetraspanins Protein kinase C
The interactions between the integrated complex array of integral and peripheral cell adhesion molecules (CAMs) are tightly controlled by kinases such as Protein Kinase C (PKC) in response to changes in external or internal forces and/or signaling. Focusing on the complex of EpCAM-claudin-tetraspanin-driven ovarian cancer, we described a sequence of events by which role of PKCs located in the tetraspanin enriched microdomains affected on the interactions and performed immunoprecipitations in PKC activator and inhibitors-treated ovarian cancer cells and xenograft ovarian cancer mouse models. Activated PKC isoforms associated with tetraspanins and induced detectable changes in the claudin phosphorylation state. These results suggest that PKC targets claudin-4 ad -7. Phosphorylation, especially by PKC δ and η of claudins was important for the interactions between claudin-4, -7 and EpCAM. These results represent the direct evidence that phosphorylation of claudins by PKCs functions in the EpCAM-claudin-tetraspanin complex formation to allow these complexes to operate in ovarian cancer progression and metastasis in vitro and in vivo.
1. Introduction During carcinogenesis, primary tumor cells lose their adhesion contacts, migrate through the lymphatic or blood system and form new tumors in the new metastasis sites. Therefore, tumor cells inevitably experience dysregulation in cell-cell adhesion, and the transformation of tumor cells is highly regulated by cell adhesion which occurs from the interactions between the different types of junctional complexes consisting of several cell adhesion molecules, cell adhesion molecules (CAMs) such as cadherins, integrins, cell surface proteoglycans, and tetraspanins on the cell surface. Type I and 39–42 kDa glycoprotein, Epithelial cell adhesion molecule (EpCAM) functions as a homophilic, epithelial-specific intercellular cell adhesion molecule [1–3] which is found expressed on a great variety of human carcinoma [4]. Also, EpCAM overexpression strongly correlates with worse overall survival, for instance in breast and ovarian cancer [5,6]. Among the most important structural and functional components of tight junctions, claudins are located in the mostly apical junctions of epithelial and endothelial cells and help to regulate paracellular permeability besides mediation to cell-to-cell adhesion. The aberrant expression of these proteins contributed to the destabilization of tight junctions and thus to
⁎
loss of cell polarity and cohesion [7–9]. In addition, tetraspanins can strongly influence cellular signaling such as adhesion, migration and invasion [10,11] by organizing laterally with other membrane proteins within the context of specialized tetraspanin-enriched microdomains (TEMs). Many studies have found correlations between tetraspanins and cancer progression [12–15]. The interactions between the integrated complex array of integral and peripheral CAMs which associate to several molecules involved in signaling cascades, as well as expressions are tightly controlled by kinases to facilitate junction assembly or disassembly. Phosphorylation acts as a switch on the cell adhesion molecules, turning “on” or “off” their interactions with other proteins. Phosphorylation which is catalyzed by kinases is a dynamic posttranslational modification and can alter protein structure, localization, protein-protein interactions and stability [16,17]. Protein Kinase C (PKC) family consists of at least 12 kinases and is involved in the phosphorylation of serine and threonine amino acid residues on the proteins. PKCs are translocated from cytoplasm to the membrane after activation by signals and play important roles in several signaling cascades ranging from cell adhesion, cellular growth, proliferation, differentiation and cell death [18,19]. Among gynecologic malignancies, ovarian cancer is the most lethal
Corresponding author at: Izmir Biomedicine and Genome Center, 35340, İzmir, Turkey. E-mail address: [email protected] (H.A. Kayali).
https://doi.org/10.1016/j.procbio.2020.01.009 Received 8 August 2019; Received in revised form 10 December 2019; Accepted 14 January 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Zehra Tavsan and Hulya Ayar Kayali, Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.01.009
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
incubated with specific primer antibodies of the studied molecules. After overnight incubation at 4 °C by rolling, the Protein A/G beads were added and incubated for 1 h at 4 °C. The immune complexes were collected by centrifuging at 14,000 rpm for 20 min 4 °C. The pellets were rinsed three times with lysis buffer. The cleared pellets were incubated in SDS-PAGE loading buffer at 95 °C for 20 min and maintained in the solubilized form by centrifuging.
as the majority will respond temporarily to surgery and cytotoxic agents and ranks fifth highest among cancer deaths for women. Due to complex and irrespective of the exact course of the pathogenesis of persistent and recurrent ovarian cancer, we previously initiated a serial analysis of the complex between cell adhesion molecules to better understand the molecular mechanisms of ovarian cancer. Co-immunoprecipitation analysis data revealed the EpCAM-claudin-4 or-7/ CD82-regulated ovarian cancer progression. From the significant body of our previous studies about the role of complex formation between EpCAM-claudin-4 or-7/CD82 in the ovarian cancer progression, better understanding of the role of this posttranslational modification, phosphorylation will provide important biologic and potentially therapeutic insights in the regulation of EpCAM-claudin-tetraspanin complexdriven ovarian cancer progression.
2.5. Western blotting After the last centrifugation step, the supernatants were mixed with SDS-PAGE loading buffer and equal amounts of total protein were loaded onto SDS-PAGE gels. SDS-PAGE was performed using a 10–12 % gel and the fractionated proteins were electroblotted onto nitrocellulose membranes. After blocking with 5 % BSA or skim milk powder in TBS with 0.1 % Tween 20 (TBST), membranes were incubated with against mono- or polyclonal antibodies of studied molecules. The membranes were washed in TBST and further incubated with HRP-conjugated secondary antibodies. The ECL Western blot analysis system was used to detect the antibodies. The density ratio of the specific bands was quantified by Image J program.
2. Materials and methods 2.1. Cell lines and cell culture The cell lines used were normal ovarian surface epithelial (OSE), A2780, OVCAR–3, SKOV–3 and A2780cis. Normal ovarian surface epithelial (OSE) cell line was purchased from Abm-Good. It was immortalized by SV–40 transfections. A2780, OVCAR–3, SKOV–3 and cisplatin-resistant A2780cis ovarian cancer cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC). The cells of passage 4 were resuscitated from liquid nitrogen stocks and cultured for less than 2 months before reinitiating culture from the same passage. ATCC, ECACC and Abm-Good had authenticated the cell lines using morphology, karyotyping, and PCR-based approaches.
2.6. Treatment of protein kinase C activator and inhibitors For studying the effects of PKCs on the interactions, 70–80 % confluent cells were incubated with 100 nm of PKC activator, PMA for 30 min at 37 °C. 5 μM of PKC δ inhibitor, rottlerin and PKC ƞ pseudosubstrate (PS) were treated for 2 h at 37 °C. 2.7. Xenograft animal studies
2.2. Cell growth conditions Six- to eight-week-old female BALB/c nude mice were provided by the Izmir Biomedicine and Genome Center (iBG) (Izmir, Turkey). The animals were housed in microisolator cages in a pathogen-free animal bio-safety level-2 facility at 22 ± 2 °C. Human SKOV-3 cells (5 × 106 cells/mice) were injected intraperitoneally (i.p.) into immunodeficient mice. Treatment started after 4 weeks of tumor formation. The mice were randomly divided into three groups (n = 6–8 per group). For the following two weeks, the mice were treated intraperitoneally with PBS, PKC ƞ PS- and rottlerin (2 times a week for 2 weeks). All procedures involving the use and care of mice were approved ethically and scientifically by the university in compliance with the Practice Guidelines for Laboratory Animals of Turkey. Formalin-fixed and paraffin-embedded tissues were sectioned (5μm-thick) and used to terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay for quantitative assessment of apoptosis. The sections were deparaffinated and dehydrated, and then TUNEL assay was carried out with an in situ Cell Death Detection Kit (Roche) following the manufacturer’s protocol [20].
A2780, OVCAR–3 and A2780cis, SKOV–3 and OSE cell lines were grown in RPMI, McCoy’s 5A and Prigrow I growth mediums, respectively. All growth mediums contain 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin. The cells were incubated in CO2 incubator, conditioned to 37 °C and 5 % CO2 levels. For in vivo injection, SKOV-3 cells in logarithmic growth phase were trypsinized and centrifuged at 800 g for 5 min, washed twice, reconstituted in PBS at a concentration of 5 × 106 cells/mice for intraperitoneal injections. 2.3. The preparation of cell and tissue lysates The cells, at confluence about 80–90 % were washed with cold phosphate buffered saline PBS. The cells were collected by cell scraper and pelleted after centrifuging at 2000 rpm for 5 min. The pellets were lysed in the lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl) including the appropriate detergent (1 % Triton X-100, Brij 96 and CHAPS) and supplemented with aprotinin, leupeptin and phenyl-methyl-sulfonylfluoride (PMSF) as proteases and phosphatases inhibitors. After sonication, the lysates were incubated in the lysis buffer on ice for 15 min and centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatants were used for western blotting and immunoprecipitation. The protein levels were determined with BCA assay. The absorbance was read at 562 nm. BSA standard was used for calibration. Total proteins were purified from snap frozen tumors after homogenization in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 % Triton X-100 in the presence of protease and phosphatase inhibitors. Lysates were cleared by centrifuging at 13,300 rpm for 20 min and protein concentration was determined using a BCA kit and continued with immunoprecipitation and western blotting.
2.8. Statistical analysis SPSS20.0 software was used to input and analyze data, measurement data comparison between two groups was by t-test and P < 0.05 indicated statistical significance in differences. 3. Results We previously initiated a serial analysis of the complex between cell adhesion molecules to better understand the molecular mechanisms of ovarian cancer using different cell lines which have isolated primary and metastatic solid tumors, and ascites fluid. Co-immunoprecipitation analysis data revealed the EpCAM-claudin-4 or-7/CD82-regulated ovarian cancer progression. At the first step of the study, we evaluated which PKC isoforms were located with tetraspanin, CD82 which formed complexes with EpCAM and claudin-4 or -7. Then, how the
2.4. Immunoprecipitation The preparation of the supernatants mentioned in the previous section. The supernatants containing about 750 μg protein were 2
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
Fig. 1. Phosphorylation modulates the interactions. (A) A2780, OVCAR-3 and SKOV-3 cells were treated with PMA and then the proteins were immunoprecipitated with the indicated antibodies. Then, the interacted PKC isoforms was confirmed by immunoblot. After the treatments with PKC η pseudosubstrate (PS) (B) and rottlerin (C) in PMA-induced A2780, OVCAR-3 and SKOV-3 cells, lysates were immunoprecipitated with the indicated antibodies. The interactions were confirmed by immunoblot.
and PKC δ in OVCAR-3 and SKOV-3 cells (Fig. 1A). In A2780 cells, the interactions between CD151 and PKC η were not determined. Further studies continued with PKC η and PKC δ among the studied PKC isoforms because the no coimmunoprecipitation with PKC α and β1. In order to investigate whether PKC changes EpCAM, claudins and tetraspanins interactions, we treated ovarian cancer cells with the PKC activator, PMA and PKC inhibitors, and examined claudin-4 and -7, EpCAM and CD82 status in claudin-4 and -7 immunoprecipitants. Fig. 1B and C showed the interactions of claudin-4 and claudin-7 with claudin-4, -7, EpCAM and CD82 after PKC η PS and rottlerin treatment compared with PMA treatment. PKC η pseudosubstrate (PS) and rottlerin specifically inhibit PKC η and δ, respectively. Without activated PKCs, immunoprecipitants of claudin-7 from A2780 cells yielded no associated claudin-4. When compared with PMA treatment, the amounts of claudin-7, -4 and CD82 were significantly decreased in claudin-4 immunoprecipitants of OVCAR-3 and SKOV-3 cells after PKC η PS treatment. Similarly, the interactions between claudin-7, -4 and CD82 with claudin-7 were diminished with PKC η inhibition. Unexpectedly, claudin-4 or claudin-7 interactions with EpCAM did not change after PKC η PS treatment. When compared with PMA treatment,
phosphorylation by PKCs affects EpCAM-claudin-4 or-7/CD82 complex was investigated by using PKC inhibitors. 3.1. The interactions of claudin are regulated by PKCs When considering regulatory mechanisms which determine assembly, remodeling/modulation, and dynamics of cell adhesion, it occurs on various levels. Interestingly, several studies demonstrated the involvement of various kinases in the phosphorylation and regulation of proteins. The phosphorylation determined the activity and interactions of a protein at the plasma membrane where numerous molecules of cell signaling pathways interact within. Starting from the consequences that PKCs associate closely with several different tetraspanin proteins upon activation and translocation, we first evaluated which PKC isoforms were located with associated tetraspanins by western blotting of PKC α, β1, δ, and η in CD82 and CD151 immunoprecipitants after activation of PKC isoforms with PMA. In the CD82 and CD151 coimmunoprecipitates of A2780, OVCAR-3 and SKOV-3 cells, PKC α and β1 were not observed (data not shown because of no bands). The CD82 and CD151 coimmunoprecipitated with PKC η 3
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
-7 by serine and threonine residues which are phosphorylated by serine and threonine kinase, PKCs compared to the cells treated with PMA and rottlerin (Fig. 3).
the amounts of claudin-4, -7 and EpCAM in the claudin-4 and claudin-7 immunoprecipitants decreased after rottlerin treatment in A2780 cells. In OVCAR-3 cells treated with rottlerin, interactions between claudin-7 and claudin-4 and, also the amounts of EpCAM in the claudin-7 immunoprecipitants were completely lost, suggesting that phosphorylated claudin-7 is the most important for the complex formation between claudin-4, -7 and EpCAM. In addition, after PKC δ inhibition with rottlerin, the claudin-4 and EpCAM amounts in claudin-4 immunoprecipitants were significantly decreased. In contrast, PKC δ inhibition decreased the amounts of claudin-4, -7 and CD82 in claudin-4 and -7 immunoprecipitants. When all results were summed, phosphorylation, especially by PKC δ and η was important for the interactions between claudin-4, -7 and EpCAM in the metastatic ovarian cell lines, OVCAR-3 and SKOV-3. Unfortunately, the ovarian cancer was diagnosed at the late stage. In addition, in accordance to the main point which is to study PKC regulation of ovarian cancer driven by EpCAMtetraspanin-claudin complex and also in the light of the recent studies of PKC δ as a prime candidate [21–23], the further studies continued with PKC δ in OVCAR-3 and SKOV-3 cells.
3.3. In vivo EpCAM-claudin-tetraspanin complex formation EpCAM, claudin-4 and -7, and CD82 coexpression correlated with the metastasis and drug resistance in ovarian cancer. To reassure the in vivo relevance of complex formation, coimmunoprecipitation of EpCAM, claudin-4 and -7, and CD82 were evaluated in the harvested tumor tissues of xenograft ovarian cancer mice. Given the absence of certain cell number on xenograft ovarian cancer models in the literature, it has been difficult to determine the cancer cell number. In our preliminary studies, we evaluated the distribution of a various number of intraperitoneal (i.p.) injected ovarian cancer cells during 4 weeks. The results in 2 × 106 cell-injected nude mice exhibited the quite small and slow-growing tumors, usually around the injection site without losing weight, proving any antisurvival effect on the nude mice. The tumors in 5 × 106 cell-injected nude mice were metastasized on the liver surface, diaphragm and colon through the peritoneal cavity. In contrast, the tumors were too large to adversely affect the survival of the animals in 10 × 106 cells-injected mice. We chose the 5 × 106 to assess the complex formation in in vivo. In line with the in vitro findings, we noted in vivo experiments that EpCAM, claudin-4 and -7, and CD82 formed a complex in TEM (Fig. 4A). Analyzing the coexpression of pairs of these molecules significantly strengthened the difference between metastatic tumors and primary tumors (data not shown). Fig. 4B showed that PKC η and δ coimmunoprecipitated with CD82 and CD151, respectively, proving the direct interaction of activated PKC with tetraspanins in TEMs. In the tumors of PKC ƞ PS- and rottlerin-treated groups, EpCAM, claudin-4 and -7, and CD82 interactions were abrogated by PKC inhibition compared to the control PMA-treated mice (Fig. 4C). To confirm the hypothesis that progression of ovarian cancer could
3.2. Claudin-4 and -7 specifically are phosphorylated by PKC δ The experiments compared to PKC activator and inhibitors treatments suggested PKC δ as a prime candidate for the phosphorylation of EpCAM and claudins, which molecules were known phosphorylated by PKCs [21,22,24]. In order to verify whether claudin-4 and -7 could be phosphorylated by PKC, ovarian cancer cells (OVCAR-3 and SKOV-3) were treated with the PKC activator and inhibitor, and phosphorylation status were examined. PKC activation induced detectable changes in the claudin phosphorylation state suggests that PKC targets claudin-4 ad -7 (Fig. 2). When claudin-4 and -7 immunoprecipitations PMA- and rottlerintreated cancer cells compared, it revealed that PKC inhibition significantly downregulated the level of phosphorylation in claudin-4 and
Fig. 2. Claudins-interacted with EpCAM and tetraspanins are phosphorylated by PKCs. PMA-activated and rottlerin-inhibited OVCAR-3 (A), SKOV-3 (B) and A2780cis (C) cells were immunoprecipitated with claudin-4 and -7 and immunoblotted for phosphothreonine and phosphoserine. 4
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
Fig. 3. Relative band intensity of phosphoserine and phosphothreonine bands of claudin-4 and -7 immunoprecipitants in PMA- and rottlerin-treated OVCAR-3 and SKOV-3 cells.
Protein Kinase C (PKC), a family of phospholipid-dependent serine/ threonine kinases plays key roles in many of the signaling pathways [25,26]. In parallel with the activation of cytosolic PKCs, they translocate to the cellular membranes. Various PKCs also associate with the specific binding proteins in the membrane [27]. However, aside from PKC interaction with the transmembrane proteoglycan syndecan-4 [28], a role for specific transmembrane proteins such as tetraspanins during PKC translocation has previously been suggested. Termini et al. showed that the translocation of PKCs to the plasma membrane leads to their specific association with tetraspanin proteins [29]. Similarly, our findings indicated that CD82 which recruited EpCAM and claudins in the TEMs was also associated with specific PKCs under PKC activation. The possible location of tetraspanin-PKC complexes in TEMs, coupled with the appearance of complexes only under mild detergent conditions, suggests weak interactions. In addition, due to translocation of PKCs to the plasma membrane in the TEMs via activation, the molecules which already constitutively associated with tetraspanin proteins become linked to PKC and these associated molecules are phosphorylated in a PKC-dependent manner. Our experiments under PKC-activated and -inhibited conditions revealed that PKC phosphorylation leads to the formation of EpCAM-claudin-tetraspanin complex. Zhang et al. indicated although formation and maintenance of tetraspanins-PKC complexes are not dependent on integrins, tetraspanins proteins can act as linker molecules, recruiting PKC into proximity with specific integrins [30]. Also, claudin-7 associated with EpCAM, CD44v6 and D6.1A and the functional tumor progression activity of EpCAM-claudin7 complex in pancreatic and colorectal carcinomas depends on claudin7 phosphorylation [31]. In the current work, we have examined the claudin-4 and -7 phosphorylation by PKC, as consequences of abrogated interactions between EpCAM, claudins and tetraspanins in PKC inhibitors-treated ovarian cancer cells. We showed that the inhibition patterns of claudin-4 and -7 phosphorylation in the presence of the PKC δ inhibitor, rottlerin which the results were consistent with the studies of D'Souza et al., indicating that phosphorylation of claudin-3 and claudin-4 by PKA and PKC ε, respectively in ovarian cancer cells [22,32]. Thus, the results provide the first evidence for the phosphorylation of endogenous claudin-4 and -7 by PKC δ in the ovarian cancer cells, and depending on the phosphorylation status, EpCAM-claudinstetraspanins complex was observed or not. Besides, the current findings were consistent with the studies that have previously shown that posttranslation modifications by phosphorylation of the overexpressed claudins may inhibit their tight junction-related function while allowing other pro-oncogenic effects. Because claudins in the tight junctions found in the lipid rafts as characterized detergent-insoluble
be stopped if the complex formation was impaired by PKC inhibition, the growth-inhibitory effects of PKC inhibitors on ovarian cancer were examined in vivo. For this purpose, i.p. xenograft ovarian cancer models were employed and the effects of PKC inhibitors on the intra-abdominal dissemination of ovarian cancer tumors, ascites formation, and tumor growth were examined. Drugs showed no apparent toxicity throughout the study. The mice treated with PMA had significant abdominal swelling because of ascites formation, whereas swelling was much less in mice treated with PBS and PKC inhibitors (data not shown). At 2 weeks after initiation of treatment, postmortem examination showed that the tumors were on the surface of the peritoneum, intestines, omentum, and uterus in the PKC inhibitors treated groups. However, in the control PMA treated groups, in addition to the peritoneum, intestines, omentum, and uterus-disseminated tumors, the tumors on the diaphragm and/or the hilus of the liver varied. Especially, the tumor distribution in rottlerin-treated mice significantly decreased compared both PMA- and PKC ƞ PS-treated groups (data not shown). In spite of the reduction of metastasis to the distant organs, tumor volumes increased significantly in the PKC inhibitor-treated group compared to PMA-treated group (Fig. 4D). Because, there was the inflammation in the tumors of PKC inhibitor-treated groups (data not shown). To gain insight into the mechanism of rottlerin-dependent tumor metastasis reduction in vivo, apoptosis was assessed in the harvested tumors from rottlerin- and PKC ƞ PS-treated mice compared to the PMA and PBS-treated groups by TUNEL analysis. As shown in Fig. 4E, there were many TUNEL-positive cells inside of the small tumors obtained on the liver and diaphragm while apoptosis started from the outer side of tumors tissue taken from rottlerin-treated mice. In addition, in PKC ƞ PS-treated mice groups, TUNEL positive cells were observed. TUNEL staining was negative in PBS- and PMA-treated groups. These results indicated that PKC inhibition induced apoptosis. However, the relation between apoptosis, proliferation and metastasis of PKC inhibitors have to be further studied. 4. Discussion Epithelial cells adhere to their neighbors and the extracellular matrix, mediated by different types of junctional complexes. These complexes are tightly controlled by kinases to facilitate junction assembly or disassembly. Phosphorylation acts as a switch on the cell adhesion molecules, turning “on” or “off” their interactions with other proteins by altering protein structure, localization, protein-protein interactions and stability. Recent studies highlight the regulation of cell adhesiondriven signaling during cancer progression and metastasis. 5
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
Fig. 4. The interactions in tumors of xenograft ovarian cancer mice. After 4 weeks, the tumours were taken from the animals and total proteins were purified from snap frozen tumors by homogenisation in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % Triton X-100. The indicated molecules were immunoprecipitated and analysed by western blotting (A). The lysates of control mice were immunoprecipitated with the indicated antibodies. Then, the interacted PKC isoforms was confirmed by immunoblotting (B). The xenograft ovarian cancer mice were treated with PMA and PKC inhibitors for 2 weeks and the lysates of snap frozen tissues were obtained as indicated in Material and method section. The proteins were immunoprecipitated with the indicated antibodies. Then, the interacted molecules were confirmed by immunoblotting (C). Appearance of tumors after treatment of mice with PKC ƞ PS and rottlerin. Four representative mice inoculated with SKOV-3 cells treated i.p. with PBS (control), PKC ƞ PS and rottlerin. Treatments were started 2 weeks after inoculation with SKOV-3 cells. At the end of the experiment (2 weeks of treatment), mice were sacrificed. At autopsy, tumors were excised and ascites fluid was collected (D). Rottlerin- and PKC η pseudosubstrate (PS) induced cell apoptosis in tumor tissues in vivo was measured by TUNEL assay compared with PMA- and PBS-treated xenograft ovarian cancer mice. The representative images are shown with TUNEL signals (green) (E) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
activity. Only when complexes are located in glycolipid-enriched membrane domains, cells have displayed increased apoptosis resistance and an increased tendency to aggregate [34]. Cell culture experiments with ovarian cancer cells which have
fraction [33], to display the pro-oncogenic functions, overexpression, complex formation with EpCAM and tetraspanins of claudins, and also complex location in the detergent-soluble glycolipid-enriched membrane domains outside of tight junctions is crucial for its functional 6
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
different character showed that coexpression of EpCAM, claudins and tetraspanin in the glycolipid-enriched microdomains, TEMs was related to ovarian cancer progression and also cisplatin resistance. Thus, we asked whether the coexpression of these markers might provide a diagnostic and/or prognostic variable. In line with our hypothesis that to mimic metastatic stage in ovarian cancer patients, we generated xenograft ovarian cancer model by intraperitoneal injection of metastatic SKOV-3 cells. The immunoprecipitation experiments of xenograft tumors demonstrated similar results with cell line experiments. Similarly, in the experiments of Schmidt et al. and Ladwein et al., the metastasizing gastrointestinal tumors of the rat frequently express a complex that includes the tetraspanin D6.1A, EpCAM, claudin-7, and CD44v6 [31,34]. The evaluation of colorectal cancer and liver metastasis patients, as well as tumor-free colon and liver tissue showed that coexpression and complex formation of the EpCAM, claudin-7, CO-029, and CD44v6 molecules was correlated with clinical variables and apoptosis resistance [35]. The involvement of EpCAM, CD44v6 and claudin-7 in the progression of thyroid cancer from an indolent to an aggressive phenotype has been showed [36]. In accordance with our findings, PKC inhibitor, chelerythrine treatment of nude mice bearing HNSCC resulted in significant tumor growth delay [37]. Also, combined inhibition of PKC and histone deacetylase 1 lowered drug doses needed in vitro, in vivo, and ex vivo in patient-derived advanced basal cell carcinomas explants [38]. In whole lung tumors, fluorescence microscopy quantified double-positive green/red tumor cells experiencing caspase cleavage after treatment with PKC inhibitors, proving that PKC activation was significantly linked to the survival of highly metastatic cells [39]. Spleen and liver tissue sections of BALB/c mice treated with thymoquinone showed an increased number of apoptotic cells [40]. Collectively, we showed a novel mechanism which is responsible for ovarian cancer progression in vitro and in vivo. Our findings assigned PKC-modulated EpCAM-claudin-4/-7-CD82 complex. The disruption of these complex as a result of PKC inhibition or different approaches is a good candidate to stop or slow down ovarian cancer invasion, metastasis and drug resistance.
[3]
[4]
[5]
[6]
[7] [8] [9]
[10] [11] [12] [13]
[14]
[15]
[16]
[17]
[18]
Funding
[19]
This work was supported by TUBITAK (The Scientific and Technological Research Council of Turkey-113Z641 project number).
[20]
Author statement [21]
Conception and design: H. Ayar Kayali Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z. Tavsan Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Tavsan Writing, review, and/or revision of the manuscript: H. Ayar Kayali and Z. Tavsan
[22]
[23]
[24]
Declaration of Competing Interest
[25]
No potential conflicts of interest were disclosed.
[26]
Acknowledgements [27]
We gratefully thank the Graduate School of Natural and Applied Sciences, and İzmir Biomedicine and Genome Center.
[28]
References
[29]
[1] P.A. Baeuerle, O. Gires, EpCAM (CD326) finding its role in cancer, Br. J. Cancer 96 (2007) 417–423, https://doi.org/10.1038/sj.bjc.6603494. [2] M. Balzar, I.H. Briaire-de Bruijn, H.A.M. Rees-Bakker, F.A. Prins, W. Helfrich, L. de Leij, G. Riethmuller, S. Alberti, S.O. Warnaar, G.J. Fleuren, S.V. Litvinov, Epidermal
[30]
[31]
7
growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions, Mol. Cell. Biol. 21 (2001) 2570–2580, https:// doi.org/10.1128/MCB.21.7.2570-2580.2001. S.V. Litvinov, M.P. Velders, H.A.M. Bakker, G.J. Fleuren, S.O. Warnaar, Ep-CAM: A human epithelial antigen is a homophilic cell-cell adhesion molecule, J. Cell Biol. 125 (1994) 437–446, https://doi.org/10.1083/jcb.125.2.437. L. Huang, Y. Yang, F. Yang, S. Liu, Z. Zhu, Z. Lei, J. Guo, Functions of EpCAM in physiological processes and diseases (Review), Int. J. Mol. Med. 42 (2018) 1771–1785, https://doi.org/10.3892/ijmm.2018.3764. T. Hiraga, S. Ito, H. Nakamura, EpCAM expression in breast cancer cells is associated with enhanced bone metastasis formation, Int. J. Cancer 138 (2016) 1698–1708, https://doi.org/10.1002/ijc.29921. J. Zheng, L. Zhao, Y. Wang, S. Zhao, M. Cui, Clinicopathology of EpCAM and EGFR in human epithelial ovarian carcinoma, Open Med. 12 (2017) 39–44, https://doi. org/10.1515/med-2017-0007. S. Tabariès, P.M. Siegel, The role of claudins in cancer metastasis, Oncogene 36 (2017) 1176–1190, https://doi.org/10.1038/onc.2016.289. M.J. Kwon, Emerging roles of claudins in human cancer, Int. J. Mol. Sci. 14 (2013) 18148–18180, https://doi.org/10.3390/ijms140918148. S. Tabariès, A. McNulty, V. Ouellet, M.G. Annis, M. Dessureault, M. Vinette, Y. Hachem, B. Lavoie, A. Omeroglu, H.G. Simon, L.A. Walsh, S. Kimbung, I. Hedenfalk, P.M. Siegel, Afadin cooperates with claudin-2 to promote breast cancer metastasis, Genes Dev. 33 (2019) 180–193, https://doi.org/10.1101/gad. 319194.118. C.M. Termini, J.M. Gillette, Tetraspanins function as regulators of cellular signaling, Front. Cell Dev. Biol. 5 (2017), https://doi.org/10.3389/fcell.2017.00034. X. Jiang, J. Zhang, Y. Huang, Tetraspanins in cell migration, Cell Adhes. Migr. 9 (2015) 406–415, https://doi.org/10.1080/19336918.2015.1005465. M.E. Hemler, Tetraspanin proteins promote multiple cancer stages, Nat. Rev. Cancer 14 (2014) 49–60, https://doi.org/10.1038/nrc3640. F.Q. Hou, X.F. Lei, J.L. Yao, Y.J. Wang, W. Zhang, Tetraspanin 1 is involved in survival, proliferation and carcinogenesis of pancreatic cancer, Oncol. Rep. 34 (2015) 3068–3076, https://doi.org/10.3892/or.2015.4272. J. Lu, J. Li, S. Liu, T. Wang, A. Ianni, E. Bober, T. Braun, R. Xiang, S. Yue, Exosomal tetraspanins mediate cancer metastasis by altering host microenvironment, Oncotarget 8 (2017) 62803–62815, https://doi.org/10.18632/oncotarget.19119. R.R. Malla, S. Pandrangi, S. Kumari, M.M. Gavara, A.K. Badana, Exosomal tetraspanins as regulators of cancer progression and metastasis and novel diagnostic markers, Asia J. Clin. Oncol. 14 (2018) 383–391, https://doi.org/10.1111/ajco. 12869. K. Aoki, K. Yoshida, Biological consequences of priming phosphorylation in cancer development, Protein Phosphorylation (2017), https://doi.org/10.5772/ intechopen.70039. F. Ardito, M. Giuliani, D. Perrone, G. Troiano, L. Lo Muzio, The crucial role of protein phosphorylation in cell signalingand its use as targeted therapy (Review), Int. J. Mol. Med. 40 (2017) 271–280, https://doi.org/10.3892/ijmm.2017.3036. A.C. Newton, Protein kinase C: perfectly balanced, Crit. Rev. Biochem. Mol. Biol. 53 (2018) 208–230, https://doi.org/10.1080/10409238.2018.1442408. A. Tarafdar, A.M. Michie, Protein kinase C in cellular transformation: A valid target for therapy? Biochem. Soc. Trans. 42 (2014) 1556–1562, https://doi.org/10.1042/ BST20140255. C. Pu, S. Chang, J. Sun, S. Zhu, H. Liu, Y. Zhu, Z. Wang, R.X. Xu, Ultrasoundmediated destruction of LHRHa-targeted and paclitaxel-loaded lipid microbubbles for the treatment of intraperitoneal ovarian cancer xenografts, Mol. Pharm. 11 (2014) 49–58, https://doi.org/10.1021/mp400523h. N. Maghzal, H.A. Kayali, N. Rohani, A.V. Kajava, F. Fagotto, EpCAM controls actomyosin contractility and cell adhesion by direct inhibition of PKC, Dev. Cell 27 (2013) 263–277, https://doi.org/10.1016/j.devcel.2013.10.003. T. D’Souza, F.E. Indig, P.J. Morin, Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells, Exp. Cell Res. 313 (2007) 3364–3375, https://doi.org/10.1016/j.yexcr.2007.06.026. S. Jaken, P.J. Parker, Protein kinase C binding partners, Bioessays 22 (2000) 245–254, https://doi.org/10.1002/(SICI)1521-1878(200003)22:3<245::AIDBIES6>3.0.CO;2-X. C.M. Van Itallie, J.M. Anderson, Phosphorylation of tight junction transmembrane proteins: many sites, much to do, Tissue Barriers 6 (2018), https://doi.org/10. 1080/21688370.2017.1382671. G. Chandrika, K. Natesh, D. Ranade, A. Chugh, P. Shastry, Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling, Sci. Rep. 6 (2016), https://doi.org/10.1038/srep22455. S.M.A. Islam, R. Patel, M. Acevedo-Duncan, Protein Kinase C-ζ stimulates colorectal cancer cell carcinogenesis via PKC-ζ/Rac1/Pak1/β-Catenin signaling cascade, Biochim. Biophys. Acta - Mol. Cell Res. 1865 (2018) 650–664, https://doi.org/10. 1016/j.bbamcr.2018.02.002. S.J. Van Deventer, V.M.E. Dunlock, A.B. Van Spriel, Molecular interactions shaping the tetraspanin web, Biochem. Soc. Trans. 45 (2017) 741–750, https://doi.org/10. 1042/BST20160284. E.S. Oh, A. Woods, J.R. Couchman, Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C, J. Biol. Chem. 272 (1997) 8133–8136. C.M. Termini, K.A. Lidke, J.M. Gillette, Tetraspanin CD82 Regulates the spatiotemporal dynamics of PKCα in acute myeloid leukemia, Sci. Rep. 6 (2016), https:// doi.org/10.1038/srep29859. S. Hwang, T. Takimoto, M.E. Hemler, Integrin-independent support of cancer drug resistance by tetraspanin CD151, Cell. Mol. Life Sci. 76 (2019) 1595–1604, https:// doi.org/10.1007/s00018-019-03014-7. D.S. Schmidt, P. Klingbeil, M. Schnölzer, M. Zöller, CD44 variant isoforms associate
Process Biochemistry xxx (xxxx) xxx–xxx
Z. Tavsan and H.A. Kayali
[32]
[33]
[34]
[35]
[36]
with tetraspanins and EpCAM, Exp. Cell Res. 297 (2004) 329–347, https://doi.org/ 10.1016/j.yexcr.2004.02.023. T. D’Souza, R. Agarwal, P.J. Morin, Phosphorylation of Claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells, J. Biol. Chem. 280 (2005) 26233–26240, https://doi.org/10. 1074/jbc.M502003200. A. Nusrat, C.A. Parkos, P. Verkade, C.S. Foley, T.W. Liang, W. Innis-Whitehouse, K.K. Eastburn, J.L. Madara, Tight junctions are membrane microdomains, J. Cell. Sci. 113 (2000) 1771–1781. M. Ladwein, U.F. Pape, D.S. Schmidt, M. Schnölzer, S. Fiedler, L. Langbein, W.W. Franke, G. Moldenhauer, M. Zöller, The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7, Exp. Cell Res. 309 (2005) 345–357, https://doi.org/10.1016/j.yexcr.2005.06.013. S. Kuhn, M. Koch, T. Nübel, M. Ladwein, D. Antolovic, P. Klingbeil, D. Hildebrand, G. Moldenhauer, L. Langbein, W.W. Franke, J. Weitz, M. Zöller, A complex of EpCAM, claudin-7, CD44 variant isoforms, and tetraspanins promotes colorectal cancer progression, Mol. Cancer Res. 5 (2007) 553–567, https://doi.org/10.1158/ 1541-7786.MCR-06-0384. T. Okada, T. Nakamura, T. Watanabe, N. Onoda, A. Ashida, R. Okuyama, K.I. Ito, Coexpression of EpCAM, CD44 variant isoforms and claudin-7 in anaplastic thyroid
[37]
[38]
[39]
[40]
8
carcinoma, PLoS One 9 (2014) 1–13, https://doi.org/10.1371/journal.pone. 0094487. S.J. Chmura, M.E. Dolan, a Cha, H.J. Mauceri, D.W. Kufe, R.R. Weichselbaum, In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo, Clin. Cancer Res. 6 (2000) 737–742. A.N. Mirza, M.A. Fry, N.M. Urman, S.X. Atwood, J. Roffey, G.R. Ott, B. Chen, A. Lee, A.S. Brown, S.Z. Aasi, T. Hollmig, M.A. Ator, B.D. Dorsey, B.R. Ruggeri, C.A. Zificsak, M. Sirota, J.Y. Tang, A. Butte, E. Epstein, K.Y. Sarin, A.E. Oro, Combined inhibition of atypical PKC and histone deacetylase 1 is cooperative in basal cell carcinoma treatment, JCI Insight 2 (2017), https://doi.org/10.1172/jci. insight.97071. S.-H. Hong, L. Ren, A. Mendoza, A. Eleswarapu, C. Khanna, Apoptosis resistance and PKC signaling: distinguishing features of high and low metastatic cells, Neoplasia 14 (2012) 249–258, https://doi.org/10.1593/neo.111498. L.Z. Ali Salim, R. Othman, M.A. Abdulla, K. Al-Jashamy, H.M. Ali, P. Hassandarvish, F. Dehghan, M.Y. Ibrahim, F.A.E. Ahmed Omer, S. Mohan, Z. Wang, Thymoquinone inhibits murine leukemia WEHI-3 Cells in vivo and in vitro, PLoS One 9 (2014) e115340, , https://doi.org/10.1371/journal.pone.0115340.