Bhushan Thakur

Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells.

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.

p53 Loses grip on PIK3CA expression leading to enhanced cell survival during platinum resistance

Tumour suppressor p53, a master transcriptional regulator determines cell fate through preferential activation/repression of a myriad of genes during stress. Till date, activation and preferential binding of p53 on different promoters was reported to be influenced by the nature, strength and duration of stress which mediates its post translational modifications. Cisplatin, a widely used cytotoxic drug represses PIK3CA promoter activity and attenuates PI3K/AKT cell survival pathway through p53 activation in sensitive cells. However, very little is understood about the overall mechanism of p53‐PIK3CA interaction and influence of p53 on the transcriptional status of PIK3CA during cisplatin resistance. Here we showed that cisplatin could dynamically alter p53 occupancy between the p53 binding sequences present in PIK3CA promoter in ovarian and breast cancer cells. This altered occupancy is dictated by higher acetylation and hyper‐phosphorylation at serine 15, serine 20 and serine 46 residues. Interestingly, cisplatin resistant cells when challenged with cisplatin demonstrated abolished PIK3CA promoter attenuation, low level of p53 binding, and loss of p53 serine 46 phosphorylation. A phosphorylation deficient S46A mutant failed to repress PIK3CA in p53 deficient cells. Elevated expression of Bcl2, P27 and cFLIP indicated a pro‐survival state in these resistant cells. Non‐invasive real time imaging using two different luciferase reporters showed that cisplatin could simultaneously induce PIK3CA attenuation and p53 activation with growth regression in sensitive tumours but not in the resistant tumours where only low level of p53 activation and sustained growth was observed. This is the first report on phosphorylation of p53 serine 46 as a modulator of p53‐PIK3CA promoter interaction which influences altered binding of p53 at different consensus sequences in the same promoter in response to chemotherapeutic stress. Absence of such modulation in resistant cellular milieu influences cellular homoeostasis in platinum‐resistant cells probably due to altered post translational modification of p53.

Tumor suppressor protein p53 exerts negative transcriptional regulation on human sodium iodide symporter gene expression in breast cancer

PurposeAberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) is well documented but the transcription factors (TF) regulating its aberrant expression is poorly known. We identify the presence of three p53 binding sites on the human NIS promoter sequence by conducting genome-wide TF analysis, and further investigate their regulatory role.MethodsThe differences in transcription and translation were measured by real-time PCR, luciferase reporter assay, site-directed mutagenesis, in vivo optical imaging, and chromatin immunoprecipitation. The relation of NIS and p53 in clinical samples was judged by TCGA data analysis and immunohistochemistry.ResultsOverexpression of wild-type p53 as a transgene or pharmacological activation by doxorubicin drug treatment shows significant suppression of NIS transcription in multiple BC cell types which also results in lowered NIS protein content and cellular iodide intake. NIS repression by activated p53 is further confirmed by non-invasive bioluminescence imaging in live cell and orthotropic tumor model. Abrogation of p53-binding sites by directional mutagenesis confirms reversal of transcriptional activity in wild-type p53-positive BC cells. We also observe direct binding of p53 to these sites on the human NIS promoter. Importantly, TCGA data analysis of NIS and p53 co-expression registers an inverse relationship between the two candidates.ConclusionOur data for the first time highlight the role of p53 as a negative regulator of functional NIS expression in BC, where the latter is a potential targeted radioiodine therapy candidate. Thus, the study provides an important insight into prospective clinical application of this approach that may significantly impact the patient with mutant versus wild-type p53 profile.

Assessing Therapeutic Potential of Magnetic Mesoporous Nanoassemblies for Chemo-Resistant Tumors.

Smart drug delivery system with strategic drug distribution is the future state-of-the-art treatment for any malignancy. To investigate therapeutic potential of such nanoparticle mediated delivery system, we examined the efficacy of dual drug-loaded, pH and thermo liable lipid coated mesoporous iron oxide-based magnetic nanoassemblies (DOX:TXL-LMMNA) in mice bearing both drug sensitive (A2780S) and drug resistant (A2780-CisR) ovarian cancer tumor xenografts. In presence of an external AC magnetic field (ACMF), DOX:TXL-LMMNA particles disintegrate to release encapsulated drug due to hyperthermic temperatures (41-45 ºC). In vivo bio distribution study utilizing the optical and magnetic properties of DOX:TXL-LMMNA particles demonstrated minimum organ specific toxicity. Noninvasive bioluminescence imaging of mice bearing A2780S tumors and administered with DOX-TXL-LMMNA followed by the application of ACMF revealed 65% less luminescence signal and 80% mice showed complete tumor regression within eight days. A six months follow-up study revealed absence of relapse in 70% of the mice. Interestingly, the A2780-CisR tumors which did not respond to drug alone (DOX:TXL) showed 80% reduction in luminescence and tumor volume with DOX:TXL-LMMNA after thermo-chemotherapy within eight days. Cytotoxic effect of DOX:TXL-LMMNA particles was more pronounced in A2780-CisR cells than in their sensitive counterpart. Thus these novel stimuli sensitive nanoassemblies hold great promise for therapy resistant malignancies and future clinical applications.

Soft drug-resistant ovarian cancer cells migrate via two distinct mechanisms utilizing myosin II-based contractility

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the migration mechanisms of drug-resistant cancer cells remain incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are more contractile and softer than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR cells, indicative of a protease-dependent mode of migration in CisR cells and a protease-independent mode of migration in PacR. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin II signaling pathway, regulates both modes of migration. Confined migration experiments establish the role of myosin IIA and IIB in mediating nuclear translocation and regulation of proteolytic activity. Collectively, our results highlight the importance of myosin II as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells.

Cisplatin triggers cancer stem cell enrichment in platinum-resistant cells through NF-κB-TNFα- PIK3CA loop

BackgroundParallel to complex alteration in molecular and cellular events, enrichment of cancer stem cells (CSC) contributes significantly in deliberation and maintenance of cisplatin resistance. Cisplatin mediated CSC enrichment is well established in various cancers, yet the underlying mechanism is largely unknown. Cisplatin also promotes transcriptional upregulation of PIK3CA, hence activating PI3K/AKT signaling in resistant cells. However, such cisplatin-induced transcriptional regulators of PIK3CA and their impact on cancer stem cell population in resistant cells are largely unknown.MethodsDNA-binding protein pulldown using PIK3CA promoter as bait followed by nLCMS was used to identify, cisplatin-induced potential transcriptional regulators of PIK3CA promoter. PIK3CA promoter activity was estimated by luciferase based reporter assay. ChIP was used to assess interaction of NF-κB with PIK3CA promoter. CSC-enriched side-population was sorted using DCV-dye exclusion methods. All the gene expression levels were assessed using qPCR.ResultsUsing a transcription factor pull-down assay with PIK3CA promoter, we identified NF-κB as a prime regulator, which escalates both TNFα and PIK3CA expression only in CSC enriched side-population (SP) but not in non side-population (NSP) in platinum resistant ovarian cancer cells upon cisplatin treatment. This SP-specific NF-κB-TNFα-PIK3CA bi-modal loop, on one hand, maintains persistent activation of NF-κB through TNFα- NF-κB autocrine loop, while NF-κB-PIK3CA loop nurture CSC population under cisplatin treatment. Activation of PI3K/AKT signalling drives SP’s into an undifferentiated, anti-apoptotic stage through upregulating P21, P27,cFLIP expression. Contrarily, lack of active NF-κB-TNFα-PIK3CA loop makes NSPs vulnerable towards cisplatin and undergoes apoptosis. Altogether, cisplatin enriches cancer stem cells properties in SP fraction, which is evident from increased levels of pluripotency gene OCT4/SOX2/NANOG expression. Disruption of PIK3CA-NF-κB loop by Wortamannin reduces SP fraction by 1.4–1.6 fold in control and treated cells.ConclusionTogether, our study signifies an active role of NF-κB-TNFα-PIK3CA bi-modal loop in cisplatin-mediated promotion and maintenance of CSC-like population in platinum-resistant cells.

Dual Modality Imaging of Promoter Activity as a Surrogate for Gene Expression and Function

Molecular functional imaging with optical reporter genes (both bioluminescence and fluorescence) is a rapidly evolving method that allows noninvasive, sensitive, real-time monitoring of many cellular events in live cells and whole organisms. These reporter genes with optical signatures when expressed from gene-specific promoters or Cis/Trans elements mimic the endogenous expression pattern without perturbing cellular physiology. With advanced recombinant molecular biology techniques, several strategies for optimal expression from constitutive or inducible, tissue-specific and weak promoters have been developed and used for dynamic and functional imaging. In this chapter, we provide an overview of the applications of this powerful technology for imaging gene expression in living cells and rodent models.

Soft drug-resistant ovarian cancer cells invade via two distinct mechanisms utilizing myosin IIB

The failure of chemotherapeutic drugs in treatment of various cancers is attributed to the acquisition of drug resistance. However, the invasion mechanisms of drug-resistant cancer cells remains incompletely understood. Here we address this question from a biophysical perspective by mapping the phenotypic alterations in ovarian cancer cells (OCCs) resistant to cisplatin and paclitaxel. We show that cisplatin-resistant (CisR), paclitaxel-resistant (PacR) and dual drug-resistant (i.e., resistant to both drugs) OCCs are softer and more contractile than drug-sensitive cells. Protease inhibition suppresses invasion of CisR cells but not of PacR and dual cells, suggesting protease-dependent mode of invasion in CisR cells and protease-independent mode in PacR and dual cells. Despite these differences, actomyosin contractility, mediated by the RhoA-ROCK2-Myosin IIB signaling pathway regulates both modes of invasion. Myosin IIB modulates matrix metalloproteinase-9 (MMP-9) secretion in CisR cells and nuclear squeezing in PacR and dual cells, thereby highlighting its importance as a potential therapeutic target for treatment of drug-resistant ovarian cancer cells. Financial Support Authors acknowledge financial support from IIT Bombay Healthcare Initiative, CSIR andDepartment of Biotechnology (Govt. of India) (Grant # BT/PR14658/MED/31/107/2010).AK and BTwere supported by fellowships from UGC and CSIR respectively (Govt. of India). Authors declare no competing financial interests Author contributions: AK, PR and SS designed the experiments. AK performed most of the experiments and analyzed the data.BT and SG developed drug resistant cell lines and did RT-PCR. MM performed western blot. AD performed AFM experiments. SD performed gelatin zymography. ABB, PM, AM and AD helped with live cell imaging experiments. AK and SS wrote the manuscript. All authors read and approved the final manuscript. Summary statement: This study identifies drug-specific differences in the modes of invasion utilized by ovarian cancer cells, and demonstrates the role of myosin IIB in regulating both modes of invasion.