Background
Chemotherapy is one of the prevailing methods to manage neoplastic growth. Unfortunately, generation of resistance substantially handicaps the efficacy and results in significant mortality in cancer patients. While molecular alteration in signaling cascades aid in acquirement and maintenance of resistance, a small fraction of inherently resistant cancer stem cells (CSC) help in repopulating the chemoresistant tumor [
1‐
3]. In particular, these drug resistant CSC’s evolve to resist therapy setbacks resulting in incessant growth and relapse. Hence targeting the CSC component has a great therapeutic potential in therapy-resistant disease. However, theoretically achievable, such objective is extremely challenging and requires in depth understanding of how CSC’s response towards chemotherapeutics.
CSC’s are a small fraction of heterogeneous tumor population identified by surface markers like CD34+/CD38- (AML), ESA+/CD44+/CD24-(low) (breast cancer) and functional properties such as side-population (SP) or aldehyde dehydrogenase activity [
1]. Though association between CSCs and chemoresistance is well established, the key molecular events involved in the regulation of CSCs remain largely unknown. Till date, differential activation of PI3K/AKT, WNT, NOTCH and NF-κB signaling are linked to maintenance of CSC phenotype and chemoresistance [
4‐
6]. However, the actions and the outcomes of cancer therapeutics on signaling cascades in CSCs still remain poorly understood.
Cisplatin, a DNA damaging agent, also modulates several signalling cues including c-ABL, p53 signaling, MAPK/JNK/ERK, PI3K/AKT, NF-κB-signalling, FAK and WNT-signaling [
7]. Cisplatin resistance is a net effect of multiple mechanisms that either inhibit apoptosis, promote cell survival, or both. Amongst these, the nuclear Factor-kappa B (NF-κB) has been identified as a key player in platinum resistance [
8,
9]. A variety of stimuli coalesces on NF-κB activation, which mediated upregulation of
cFLIP,
BCl-XL, XIAP and favours survival of cisplatin resistant cells [
8,
9]. NF-κB also prevents cisplatin mediated histone acetylation and BRCA1 nuclear translocation in HNSCC and inhibit cisplatin cytotoxicity [
10]. In response to extracellular signals, a number of RTKs can activate NF-κB via PI3K/AKT or JNK/STAT pathway [
11]. In many cancers, hyperactivation of PI3K/AKT, a key survival pathway contributes to tumor growth, metastatic competence, and therapy-resistance [
12]. Besides RTK mediated activation, PI3K gene is regulated by few central transcription factors (p53, NF-κB, FOXO3a, YB1) in chemosensitive cells [
13], however, regulators of
PIK3CA in chemoresistant cells particularly in response to drugs has never been identified. We have been investigating the underlying mechanism of upregulated PI3K expression in platinum-resistant cells, and demonstrated that in absence of a cisplatin-induced Ser46-phosphorylation, p53 failed to bind and represses
PIK3CA promoter leading to activation of PI3K/AKT signalling that actively sustained survival but not proliferation of resistant cells [
14]. Though lack of PI3K promoter attenuation by cisplatin in resistant cells is indicative of loss of p53’s repressive action, it does not explain the observed increase in
PIK3CA expression and points toward a second level of regulation for this critical gene and associated signalling.
In the present study, we identified cisplatin responsive potential transcriptional activators of PIK3CA in resistant cells and explored the consequence of this intricate regulation in preserving resistance and CSC-characteristics. Through DNA-protein pulldown, NF-κB was identified as a key cisplatin responsive transcription factor, which escalated PIK3CA specifically in CSC-enriched SP cells and governed an anti-apoptotic, dormant state. Lack of drug-induced quiescence in non-CSC fraction attributed to their susceptibility towards cisplatin. Cisplatin induced an intricate bi-modal feedback loop between TNFα-NF-κB & NF-κB-PI3K signalling leading to maintenance of CSC-phenotype. This is probably the first mechanistic report on how cisplatin drives enrichment of CSCs in platinum-resistant cells thereby favouring a tilt towards resistance maintenance.
Discussion
Chemoresistance, either intrinsic or acquired, substantially handicaps the efficacy of chemotherapy, escalating mortality rates in cancer patients. Acquirement of resistance towards chemotherapeutics is a dynamic and multifactorial process and influenced by cancer stem cell (CSC) enrichment [
3,
21,
22]. Though platinum resistance is a common problem for cervical, head and neck, and non-small-cell lung cancer management, it is particularly devastating for epithelial ovarian cancer (EOC) patients as 50% of the therapy-responders ultimately show chemoresistant-relapse [
7,
23]. Utilizing A2780-cisplatin resistant cellular model and two naturally occurring cisplatin resistant cells (TOV21G and SKOV3) [
14], we attempted to address a unique question of how cisplatin impact existing CSCs in resistant cells. In all these cells, cisplatin enhanced the inherently drug resistant side-population with increased
PIK3CA expression. Activation of PI3K/AKT pathway is known to favour cellular endurance against chemotherapeutics and maintenance of CSC-population [
2,
18] but regulators of this pathway in drug-resistant cells are yet to be identified. Using transcription-factor pulldown assay, we identified NF-κB, β-catenin and CREB as cisplatin-responsive transcriptional activators of
PIK3CA promoter. Amongst them, NF-κB differentially activated
PIK3CA only in CSC-enriched side-population but not in non-SP. Both cisplatin and TNFα induced interaction between NF-κB and
PIK3CA promoter. Further, inhibition of PI3K-activity dramatically reduced this CSC-enriched SP fraction. Enhanced nuclear co-localization of NF-κB with OCT4 and augmented expression of
OCT4,
SOX2 and
NANOG in SP signified enrichment of CSC properties by cisplatin. Finally, gene expression analysis revealed that cisplatin mediated activation of PI3K/AKT led to an anti-apoptotic, dormant stage, which aided CSC to evade therapeutic action while actively proliferating non-CSC cells succumbed to cisplatin’s action. Intriguingly, TNFα, a well-known activator of NF-κB and an NF-κB–regulated gene showed enhanced expression following cisplatin treatment only in SP cells. Collectively, our data emphasize that therapeutic intervention to platinum-resistant ovarian cancer cells favours a predominant quiescent state in SP via an interdependent positive feedback loop between TNFα-NF-κB and PI3K/AKT signalling.
Cisplatin forms the first line therapy against malignancies including ovarian, breast and colorectal cancer, however, acquirement of resistance impaired therapeutic efficacy [
6,
7]. Amongst several molecular determinants of cisplatin resistance, activated PI3K/AKT pathway turns out to be a key signalling that also aids in survival of CSCs [
1,
2]. Lee et al., (2005) showed that escalated
PIK3CA expression in OVCAR-3/CDDP (resistant) cells leads to inhibition of
BAX translocation via activated PI3K/AKT conforming platinum-resistance [
24]. Amplification of
PIK3CA was associated with ovarian and uterine cervical carcinoma, which led to active PI3K/AKT signaling and resistance acquirement [
25,
26]. However, mechanism for augmented
PIK3CA expression is less dissected at molecular level. Intriguingly, we observed cisplatin itself could upregulate
PIK3CA expression in resistant but not in sensitive cells [
14]. Several transcription-factors (TF) such as p53/p73, cMyc, YB-1, CTF-2, ATF-4, ZNF143, mTFA, AP1, NF-κB, OCT1, SP1, β-catenin and CREB are known to be associated with cisplatin resistance, regulating expression of resistance associated genes [
27,
28]. Among all these TFs, cisplatin directly influences transcriptional activities of p53, YB-1, ZNF143, mTFA, NF-κB, ATF-2, 3 and 4 [
27]. We earlier reported that cisplatin-mediated phosphorylation of p53 at Ser46 promotes its binding, and repress
PIK3CA promoter in sensitive cells. Absence of such phosphorylation in cisplatin-treated resistant cells resulted in loss
PIK3CA attenuation [
14] which, however, does not explain the apparent increase in
PIK3CA expression level. Till now, only YB-1, NF-κB and FOXO3a are known to induce
PIK3CA expression in unstressed condition [
13]. No information is available on regulators of
PIK3CA modulation in platinum-resistant cells in response to cisplatin. Thus to identify cisplatin-responsive regulators of
PIK3CA promoter, we subjected
PIK3CA promoter bound nuclear fraction (potential transcription-factors) from untreated and cisplatin treated A2780-CisR cells to nLC-MS. We used a short OS4-
PIK3CA promoter fragment due to: A) it shows augmented promoter activity upon cisplatin treatment, B) Contains binding sites for p53, and other transcription-factors & C) to avoid multi-target binding complexity possible for full-length (1 kb) promoter. Multiple putative candidate proteins bound to
PIK3CA promoter in untreated (863 and 339) and cisplatin treated (312 and 246) A2780-CisR cells were identified which were further classified based upon their transcription associated functioning. Among the binders present exclusively in cisplatin treated samples (36%), we selected the top three
PIK3CA binders (NF-κB, β-catenin and CREB) for further validation. All these three transcription-factors are known to influence cisplatin resistance in various cancer cells. Li et al., (2016) demonstrated that cisplatin-mediated increase in β-catenin expression aids in resistance-development in oral squamous cell carcinoma [
25,
29]. While CREB is shown to be activated by cisplatin in ovarian cancer cells [
28] and hyperactivation of NF-κB is associated with cisplatin resistant human epidermoid carcinoma, KCP-4 cells [
30]. Our earlier report also demonstrates that in absence of TLR4/MyD88 signalling, ovarian cancer chemoresistance is maintained by NF-κB transcriptional activation under cisplatin treatment [
20]. Augmented
PIK3CA expression in platinum resistant H460 cells was also supported with increased NF-κB and β-catenin but not in CREB levels compared to their parental counterpart [
31]. In accordance with this, we observed augmented
PIK3CA promoter activity only in platinum resistant cells upon treatment with TNFα (NF-κB inducer) or LiCl (β-catenin inducer), while, forksolin (CREB inducer) increased
PIK3CA promoter activity in both sensitive and resistant cells. Though forskolin increased
PIK3CA expression in A2780-CisR cells, its treatment to A2780 sensitive cells led to reduction in
PIK3CA transcript levels. Other than CREB, forskolin is known to induce binding of Ets-2, phospho-p53 and AP1 TFs to MMP-2 promoter [
32]. Hence, observed decrease in
PIK3CA expression in A2780 cells may be influenced by other forskolin-induced TFs such as p53. Till date NF-κB’s role as a positive regulator for
PIK3CA promoter in ovarian cancer cells by TNFα is known [
33]. Herein, we demonstrate a unique mechanism of
PIK3CA transcriptional upregulation by NF-κB upon cisplatin, a cytotoxic drug treatment in platinum-resistant cells. ChIP assay showed that NF-κB-
PIK3CA promoter interaction increased several fold following TNFα or cisplatin treatment in A2780-CisR, TOV21G, and SKOV3 cells. Our data also suggest β-catenin complex to be another regulator of
PIK3CA promoter, however, presence of only two interacting (SMAD3 & TCF4) partners of this complex among large numbers of identified binders and minimal (~1.5) fold increase in
PIK3CA expression by LiCl did not suggest a robust role of this complex. Further experimental studies are required to elucidate the exact contribution of β-catenin complex.
Two distinct observations that cisplatin mediates SP-enrichment and increased NF-κB-
PIK3CA promoter interaction, prompted us to assess the expression level of
PIK3CA in MP, SP, and NSP fractions. Surprisingly, augmented
PIK3CA expression in response to cisplatin found only in SP but not in NSP cells. Next, for comprehensive understanding of what regulates
PIK3CA in SP, we treated SP cells with inducers of NF-κB, β-catenin or CREB. Among them, only TNFα augmented
PIK3CA promoter activity in SP but not in NSP cells, indicating NF-κB as distinct activator of
PIK3CA in SP cells. Further, to verify whether SP does contain transcriptionally active NF-κB, we used a dual-stable cellular system of A2780-CisR expressing NF-κB-transcriptional reporter and
PIK3CA sensor [
20]. Cisplatin treatment resulted in strong positive correlation between NF-κB activity and
PIK3CA promoter activity only in SP but not in MP or NSP.
Nuclear factor-κB, (NF-κB) belongs to a pivotal transcription-factor family, which controls expression of diverse genes related to immune-response, survival, proliferation, angiogenesis, and metastasis. In most cells, inhibitor of κB, (IκB) regulates NF-κB transcriptional activity [
34] and dissociation of NF-κB/IκB heterodimer leads to nuclear-translocation of NF-κB where it functions as transcription-factor. The NF-κB signalling cascade converges with several cellular pathways including PI3K/AKT, where activated AKT promotes IκB degradation via phosphorylating IKKα kinase. In most tumors, NF-κB signaling is constitutively active, regulating gene induction associated with proliferation (
CYCLIN-D1&
D2,
CDK2 and c-
MYC), growth signals (
GM-CSF and
IL6), anti-apoptosis (c
FLIP, BCL2, Bcl-xL and IAPs) and angiogenesis (
VEGF, TNFα, and
IL1) [
35]. Further, ovarian CD44 + CSCs, survive the treatment of paclitaxel and carboplatin through concomitant activation of NF-κB signalling and conferring resistance against these drugs [
4]. Hence, to understand the CSC-specific activation of NF-κB in cisplatin resistant cells, we investigated co-localisation of OCT4 with NF-κB in MP, SP and NSP. ~2–3 fold increase in nuclear NF-κB+ and OCT4+ cells in only SP but not in MP or NSP upon cisplatin treatment, clearly indicating contribution of NF-κB in both CSC-homeostasis and
PIK3CA-regulation. Enhanced expression in pluripotency gene (
OCT4,
SOX2 and
NANOG) by cisplatin exclusively in SP but not in MP or NSP warranted escalation of CSC-characteristics in SP fraction. Interestingly, same drug treatment induced distinct pluripotent gene expression in A2780-CisR, TOV21G, and SKOV3 cells probably due to intercellular differences in genetic constituents. Further to inspect NF-κB mediated
PIK3CA regulation, we assessed NF-κB-
PIK3CA promoter interaction in SP and NSP cells pre and post TNFα or cisplatin treatment. Similar to MP, SP but not NSP showed enhanced occupancy of NF-κB on
PIK3CA promoter following cisplatin treatment. TNFα, however, induced NF-κB binding on
PIK3CA promoter in both SP and NSP. This seemingly contradictory NF-κB-
PIK3CA promoter interaction by two different stimuli is not surprising as NF-κB activation by UV-C or doxorubicin is known to induce a complete different set of target genes than that by TNFα and produce entirely different functional consequences [
9].
Self-renewal and differentiation are the two major characteristics of stem cells that influence and regulate organogenesis and normal development. Likewise, cancer stem cells control tumor development through self-renewal and differentiation into proliferating tumor cells. If a cytotoxic drug can enrich CSC-population in resistant cells, it would certainly affect one of these two crucial properties. Both cisplatin and TNFα were found to favour a self-renewing undifferentiated state of CSCs as evident from 1.7–2 fold enrichment of SP. In addition, our study revealed that cisplatin mediates CSC-specific activation of NF-κB, which in turn induces expression of
TNFα and
PIK3CA. This incremented TNFα is known to act as an autocrine cue in concomitant NF-κB activation [
35,
36], while activation of NF-κB escalates
PIK3CA in CSC’s. All these data led us to hypothesize that NF-κB controlled
PIK3CA expression coordinates the CSC-survival and CSC-plasticity under the influence of a chemotherapeutics. Indeed, treating SP with wortamannin, an irreversible PI3K
-inhibitor, with and without cisplatin diminished the SP fraction. Gene expression analysis showed increased
P21,
P27 and
cFLIP and decrease in
CYCLIN-D1 and
CYCLIN-E1 in cisplatin treated SP cells pointing towards an anti-apoptotic, quiescent phase. In contrast, augmented
CYCLIN-D1&E1 expression in cisplatin treated NSP cells marking their proliferative state make them vulnerable towards drug. Activation of PI3K/AKT confers resistance against cisplatin action through up regulation of anti-apoptotic genes such as
cFLIP. Overall, CSC’s with active PI3K/AKT and NF-κB signaling acquire anti-apoptotic, quiescent state conferring survival advantage against action of chemotherapeutic drugs (Fig.
6).