Minakshi Mazumdar

Curcumin inhibits breast cancer stem cell migration by amplifying the E-cadherin/β-catenin negative feedback loop

IntroductionThe existence of cancer stem cells (CSCs) has been associated with tumor initiation, therapy resistance, tumor relapse, angiogenesis, and metastasis. Curcumin, a plant ployphenol, has several anti-tumor effects and has been shown to target CSCs. Here, we aimed at evaluating (i) the mechanisms underlying the aggravated migration potential of breast CSCs (bCSCs) and (ii) the effects of curcumin in modulating the same.MethodsThe migratory behavior of MCF-7 bCSCs was assessed by using cell adhesion, spreading, transwell migration, and three-dimensional invasion assays. Stem cell characteristics were studied by using flow cytometry. The effects of curcumin on bCSCs were deciphered by cell viability assay, Western blotting, confocal microscopy, and small interfering RNA (siRNA)-mediated gene silencing. Evaluations of samples of patients with breast cancer were performed by using immunohistochemistry and flow cytometry.ResultsHere, we report that bCSCs are endowed with aggravated migration property due to the inherent suppression of the tumor suppressor, E-cadherin, which is restored by curcumin. A search for the underlying mechanism revealed that, in bCSCs, higher nuclear translocation of beta-catenin (i) decreases E-cadherin/beta-catenin complex formation and membrane retention of beta-catenin, (ii) upregulates the expression of its epithelial-mesenchymal transition (EMT)-promoting target genes (including Slug), and thereby (iii) downregulates E-cadherin transcription to subsequently promote EMT and migration of these bCSCs. In contrast, curcumin inhibits beta-catenin nuclear translocation, thus impeding trans-activation of Slug. As a consequence, E-cadherin expression is restored, thereby increasing E-cadherin/beta-catenin complex formation and cytosolic retention of more beta-catenin to finally suppress EMT and migration of bCSCs.ConclusionsCumulatively, our findings disclose that curcumin inhibits bCSC migration by amplifying E-cadherin/beta-catenin negative feedback loop.

Inhibition of Epithelial to Mesenchymal Transition by E-cadherin Up-regulation via Repression of Slug Transcription and Inhibition of E-cadherin Degradation DUAL ROLE OF SCAFFOLD/MATRIX ATTACHMENT REGION-BINDING PROTEIN 1 (SMAR1) IN BREAST CANCER CELLS

Background: Epithelial-mesenchymal transition (EMT) is an important program in tumor metastasis. Results: SMAR1 inhibits EMT by up-regulating E-cadherin in a dual manner via repression of Slug transcription and inhibition of E-cadherin degradation. Conclusion: SMAR1 functions as a critical protein in regulating EMT. Significance: This study provides a potential mechanism for the contribution of SMAR1 in inhibiting breast cancer metastasis. The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence for which E-cadherin switch is a well-established hallmark. Discerning the molecular mechanisms that regulate E-cadherin expression is therefore critical for understanding tumor invasiveness and metastasis. Here we report that SMAR1 overexpression inhibits EMT and decelerates the migratory potential of breast cancer cells by up-regulating E-cadherin in a bidirectional manner. While SMAR1-dependent transcriptional repression of Slug by direct recruitment of SMAR1/HDAC1 complex to the matrix attachment region site present in the Slug promoter restores E-cadherin expression, SMAR1 also hinders E-cadherin-MDM2 interaction thereby reducing ubiquitination and degradation of E-cadherin protein. Consistently, siRNA knockdown of SMAR1 expression in these breast cancer cells results in a coordinative action of Slug-mediated repression of E-cadherin transcription, as well as degradation of E-cadherin protein through MDM2, up-regulating breast cancer cell migration. These results indicate a crucial role for SMAR1 in restraining breast cancer cell migration and suggest the candidature of this scaffold matrix-associated region-binding protein as a tumor suppressor.

Restoration of p53/miR-34a regulatory axis decreases survival advantage and ensures Bax-dependent apoptosis of non-small cell lung carcinoma cells.

Tumor‐suppressive miR‐34a, a direct target of p53, has been shown to target several molecules of cell survival pathways. Here, we show that capsaicin‐induced oxidative DNA damage culminates in p53 activation to up‐regulate expression of miR‐34a in non‐small cell lung carcinoma (NSCLC) cells. Functional analyses further indicate that restoration of miR‐34a inhibits B cell lymphoma‐2 (Bcl‐2) protein expression to withdraw the survival advantage of these resistant NSCLC cells. In such a proapoptotic cellular milieu, where drug resistance proteins are also down‐regulated, p53‐transactivated Bcl‐2 associated X protein (Bax) induces apoptosis via the mitochondrial death cascade. Our results suggest that p53/miR‐34a regulatory axis might be critical in sensitizing drug‐resistant NSCLC cells.

PEGylated-thymoquinone-nanoparticle mediated retardation of breast cancer cell migration by deregulation of cytoskeletal actin polymerization through miR-34a

Thymoquinone (TQ), a major active constituent of black seeds of Nigella sativa, has potential medical applications including spectrum of therapeutic properties against different cancers. However, little is known about their effect on breast cancer cell migration, which is the cause of over 90% of deaths worldwide. Herein, we have synthesized TQ-encapsulated nanoparticles using biodegradable, hydrophilic polymers like polyvinylpyrrolidone (PVP) and polyethyleneglycol (PEG) to overcome TQs poor aqueous solubility, thermal and light sensitivity as well as consequently, minimal systemic bioavailability which can greatly improve the cancer treatment efficiency. Sizes of synthesized TQ-Nps were found to be below 50 nm and they were mostly spherical in shape with smooth surface texture. Estimation of the zeta potential also revealed that all the three TQ-Nps were negatively charged which also facilitated their cellular uptake. In the present investigation, we provide direct evidence that TQ-Nps showed more efficiency in killing cancer cells as well as proved to be less toxic to normal cells at a significantly lower dose than TQ. Interestingly, evaluation of the anti-migratory effect of the TQ-Nps, revealed that PEG4000-TQ-Nps showed much potent anti-migratory properties than the other types. Further studies indicated that PEG4000-TQ-Nps could significantly increase the expression of miR-34a through p53. Moreover, NPs mediated miR-34a up-regulation directly down-regulated Rac1 expression followed by actin depolymerisation thereby disrupting the actin cytoskeleton which leads to significant reduction in the lamellipodia and filopodia formation on cell surfaces thus retarding cell migration. Considering the biodegradability, non-toxicity and effectivity of PEG4000-TQ-Nps against cancer cell migration, TQ-Nps may provide new insights into specific therapeutic approach for cancer treatment.

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways.

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.

Aspirin Suppresses the Acquisition of Chemoresistance in Breast Cancer by Disrupting an NFκB-IL6 Signaling Axis Responsible for the Generation of Cancer Stem Cells.

Acquired chemoresistance has curtailed cancer survival since the dawn of chemotherapy. Accumulating evidence suggests a major role for cancer stem cells (CSC) in chemoresistance, although their involvement in acquired resistance is still unknown. The use of aspirin has been associated with reduced cancer risk and recurrence, suggesting that the anti-inflammatory drug may exert effects on CSCs. In this study, we investigated the contribution of CSCs to acquired chemoresistance of breast cancer and the avenues for reversing such effects with aspirin. We observed that the residual risk of recurrence was higher in breast cancer patients who had acquired chemoresistance. Treatment of preexisting CSCs with a genotoxic drug combination (5-fluorouracil, doxorubicin, and cyclophosphamide) generated an NFκB-IL6-dependent inflammatory environment that imparted stemness to nonstem cancer cells, induced multidrug resistance, and enhanced the migration potential of CSCs. Treatment with aspirin prior to chemotherapy suppressed the acquisition of chemoresistance by perturbing the nuclear translocation of NFκB in preexisting CSCs. Therefore, disruptions to the NFκB-IL6 feedback loop prevented CSC induction and sensitized preexisting CSCs to chemotherapy. Collectively, our findings suggest that combining aspirin and conventional chemotherapy may offer a new treatment strategy to improve recurrence-free survival of breast cancer patients. Cancer Res; 76(7); 2000-12. ©2016 AACR.

Restoration of tumor suppressor p53 by differentially regulating pro- and anti-p53 networks in HPV-18-infected cervical cancer cells.

Abrogation of functional p53 is responsible for malignant cell transformation and maintenance of human papilloma virus (HPV)-infected cancer cells. Restoration of p53 has, therefore, been regarded as an important strategy for molecular intervention of HPV-associated malignancies. Here we report that differential regulation of pro- and anti-p53 setups not only upregulates p53 transcription but also stabilizes and activates p53 protein to ensure p53-induced apoptosis in HPV-18-infected cervical cancer. Functional restoration of p53 can be achieved by non-steroidal anti-inflammatory drug celecoxib via multiple molecular mechanisms: (i) inhibition of p53 degradation by suppressing viral oncoprotein E6 expression, (ii) promoting p53 transcription by downmodulating cycloxygenase-2 (Cox-2) and simultaneously retrieving p53 from Cox-2 association and (iii) activation of p53 via ataxia telangiectasia mutated-/p38 mitogen-activated protein kinase-mediated phosphorylations at serine-15/-46 residues. That restored p53 is functional has been confirmed by its ability of transactivating Bax and p53-upregulated modulator of apoptosis, which in turn switch on the apoptotic machinery in these cells. Studies undertaken in biopsy samples of cervical carcinoma further validated celecoxib effect. Our approaches involving gene manipulation and pharmacological interference finally highlight that celecoxib alters pro- and anti-p53 networks, not in isolation but in concert, to rejuvenate p53-dependent apoptotic program in HPV-infected cervical cancer cells.

Mithramycin A sensitizes therapy-resistant breast cancer stem cells toward genotoxic drug doxorubicin

Chemotherapy resistance is a major clinical challenge for the management of locally advanced breast cancer. Accumulating evidence suggests a major role of cancer stem cells (CSCs) in chemoresistance evoking the requirement of drugs that selectively target CSCs in combination with chemotherapy. Here, we report that mithramycin A, a known specificity protein (Sp)1 inhibitor, sensitizes breast CSCs (bCSCs) by perturbing the expression of drug efflux transporters, ATP-binding cassette sub-family G, member 2 (ABCG2) and ATP-binding cassette sub-family C, member 1 (ABCC1), survival factors, B-cell lymphoma 2 (Bcl-2) and X-linked inhibitor of apoptosis (XIAP), and, stemness regulators, octamer-binding transcription factor 4 (Oct4) and Nanog, which are inherently upregulated in these cells compared with the rest of the tumor population. In-depth analysis revealed that aberrant overexpression of Sp1 in bCSCs transcriptionally upregulates (1) resistance-promoting genes to protect these cells from genotoxic therapy, and (2) stemness regulators to sustain self-renewal potential of these cells. However, mithramycin A causes transcriptional suppression of these chemoresistant and self-renewal genes by inhibiting Sp1 recruitment to their promoters. Under such antisurvival microenvironment, chemotherapeutic agent doxorubicin induces apoptosis in bCSCs via DNA damage-induced reactive oxygen species generation. Cumulatively, our findings raise the possibility that mithramycin A might emerge as a promising drug in combinatorial therapy with the existing chemotherapeutic agents that fail to eliminate CSCs. This will consequently lead to the improvement of therapeutic outcome for the treatment-resistant breast carcinomas.

Capsaicin-Induced Activation of p53-SMAR1 Auto-Regulatory Loop Down-Regulates VEGF in Non-Small Cell Lung Cancer to Restrain Angiogenesis

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC.

Nuclear Matrix Protein SMAR1 Represses c-Fos-mediated HPV18 E6 Transcription through Alteration of Chromatin Histone Deacetylation

Background: HPV18 E6 oncogene represents one of the most promising therapeutic targets for the treatment of HPV-positive tumors. Results: Curcumin-induced SMAR1-HDAC1 recruitment at LCR and E6 region on E6 promoter deacetylates chromatin histones to attenuate c-Fos-mediated E6 transcription to reinstall p53-mediated apoptosis in HPV18-infected cervical cancer. Conclusion: SMAR1 induces E6 repression. Significance: SMAR1 is a repressor of E6-mediated anti-apoptotic network in HPV18-infected cervical cancers. Matrix attachment region (MAR)-binding proteins have been implicated in the transcriptional regulation of host as well as viral genes, but their precise role in HPV-infected cervical cancer remains unclear. Here we show that HPV18 promoter contains consensus MAR element in the LCR and E6 sequences where SMAR1 binds and reinforces HPV18 E6 transcriptional silencing. In fact, curcumin-induced up-regulation of SMAR1 ensures recruitment of SMAR1-HDAC1 repressor complex at the LCR and E6 MAR sequences, thereby decreasing histone acetylation at H3K9 and H3K18, leading to reorientation of the chromatin. As a consequence, c-Fos binding at the putative AP-1 sites on E6 promoter is inhibited. E6 depletion interrupts degradation of E6-mediated p53 and lysine acetyl transferase, Tip60. Tip60, in turn, acetylates p53, thereby restoring p53-mediated transactivation of proapoptotic genes to ensure apoptosis. This hitherto unexplained function of SMAR1 signifies the potential of this unique scaffold matrix-associated region-binding protein as a critical regulator of E6-mediated anti-apoptotic network in HPV18-infected cervical adenocarcinoma. These results also justify the candidature of curcumin for the treatment of HPV18-infected cervical carcinoma.