Tanya Das, Ph.D.

Cancer is a multifactorial disease that involves de-regulation of various signaling pathways. With an aim to develop a multiple signal modulation therapy of cancer, we have adopted different approaches, i.e., (i) to induce apoptosis, (ii) to retard metastasis and (ii) to inhibit angiogenesis in cancer cells. Moreover, since resistance to DNA damage-induced apoptosis is one of the several factors that sabotage the successful management of cancer, we also aim at regulating the cell’s decision of ‘resistance to apoptosis’ switch over and to delineate the underlying mechanisms. According to the recent hypothesis, tumorigenesis and its maintenance, metastasis and drug-resistance are driven by a limited subpopulation of tumor-initiating cells, i.e., cancer stem cells (CSCs). CSCs retain stem like properties, e.g., ability to self renew, increased proliferative capacity, and ability to differentiate into different lineages. Another aim of our study is to target these CSCs to uproot cancer from its origin. Mapping the molecular mechanisms of cancer-induced immunosuppression and immuno-editing in tumor-bearer is another aim of our research. Besides, studies on the management of cancer by molecular engineering-based therapeutic strategy, e.g., gene therapy, are also in progress.

 Objectives

 Cancer Biology

 ·        To develop a multiple signal modulation therapy of cancer: A mechanistic approach to induce apoptosis, retard metastasis and inhibit angiogenesis

·        Management of drug resistance in cancer: Targeting the problem at the molecular level

·        Integrated genome analysis to reveal oncomir/oncogene cross-talk in cancer: An approach towards a targeted therapy to uproot “root of all evils” - the cancer stem cells

·        Molecular engineering-based therapeutic strategy, e.g., gene therapy, for the management of cancer

Cancer Immunology

·        To delineate the molecular mechanisms of cancer-induced immune-suppression: An approach towards immuno-editing in tumor-bearer

Cancer Biology

To develop a multiple signal modulation therapy of cancer: A mechanistic approach to induce apoptosis, retard metastasis and inhibit angiogenesis

Cancer is a multifactorial disease that involves de-regulation of various signaling pathways. With an aim to develop a multiple signal modulation therapy of cancer, we undertook three approaches, i.e., (i) to induce apoptosis, (ii) to retard metastasis and (ii) to inhibit angiogenesis in cancer cells. Following are the results obtained so far:

The most common alterations found in breast cancer are mutation of tumor suppressor gene p53. Theaflavins induce highly resistant p53-mutated human breast cancer cell apoptosis by (i) activation of Fas-FADD-caspase-8-t-Bid pathway, and (ii) inhibition of PI-3-K-Akt-pBad survival pathway that lead to mitochondrial transmembrane potential loss, cytochrome c release and activation of executioner caspases.

Further work demonstrates that theaflavins retard human breast cancer cell migration by inhibiting NF-kB/p65 via p53–ROS feed back loop. Silencing or overexpressing the genes of interest proved that p53-dependent up-regulation of proline oxidase and down-regulation of MnSOD result in ROS generation, which activates p53 in a redox- loop via p38MAPK to block NF-kB/p65 nuclear translocation and downregulation of MMP-2 and MMP-9.

These phytochemicals also block tumor angiogenesis by reducing VEGF in breast cancer cells by (1) SMAR1-induced transcriptional inhibition of HIF-1a, the prime transcriptional factor of VEGF, and (2) by SMAR1-dependent activation of p53 that in turn induces ubiquitination and degradation of HIF-1a. Reduction in VEGF down-regulates p38 MAP kinase, an activator of HIF-1, thereby working in a feed-back loop to finally inhibit angiogenesis.

Management of drug resistance in cancer: Targeting the problem at the molecular level

Resistance to DNA damage-induced apoptosis is one of the several factors that sabotage the successful management of cancer. Results from our laboratory depict that ATM, under the influence of cellular ROS, functions as a differential switch between ‘die and not-to-die’ signals in resistant non-small cell lung cancer cells by reducing the expression of anti-apoptotic proteins, BCl-2, XIAP and surviving, while inducing the expression of pro-apoptotic proteins, p53 and Bax. Work is in progress to map the detail mechanism.

Integrated genome analysis to reveal oncomir/oncogene cross-talk in cancer: An approach towards a targeted therapy to uproot “root of all evils” - the cancer stem cells

Accumulating evidence indicates that a sub-population of cancer cells with stem-like properties, termed cancer stem cells (CSCs), exist in many different kinds of malignancies, which have a pivotal role in tumorigenesis, tumor progression, metastasis and post-treatment relapse. CSCs retain stem like properties, e.g., ability to self renew, increased proliferative capacity, and ability to differentiate into different lineages. However, how the stem-like properties of CSCs are regulated remains obscure. A recent study showed that numerous miRNAs contribute to oncogenesis (1-6) because they can function either as tumour suppressors (as is the case for miR-15a and miR-16-1) or oncogenes. Moreover, there exist functionally integrated oncomir/oncogene clusters that are frequently modified in cancer. The net effect of this modification is the differential regulation of apoptotic and survival pathways depending on the nature of the miRNA. In fact, reduction of miRNAs, let-7 and mir-30, contributes to the maintenance of the self-renewal capacity and undifferentiated status of breast cancer stem cells while enforced constitutive expression of these miRs inhibits their self-renewal capacity and induces apoptosis. However, there is dearth of information regarding the complete profile of miRNAs, their expression, upregulation and down regulation as well as their contribution in regulating different oncoproteins in tumor initiating cancer stem cells in relation to the development, metastasis and resistance of breast cancer.  Since miRNAs can be utilized as ‘one bomb multiple targets’, effects of single miRNA manipulation may have immense significance in modulating various signaling pathways to finally induce death program in resistant CSCs. Work is in progress to delineate the mechanism of oncomir/oncogene cross-talk, if any, and to target relevant miRNAs by different phytochemicals and/or synthetic compounds with an aim to uproot cancer from its origin, i.e., cancer stem cells.

Molecular engineering-based therapeutic strategy, e.g., gene therapy, for the management of cancer  

The clinical outcome for cancer remains discouraging despite efforts to optimize treatment using conventional modalities including surgery, radiotherapy and chemotherapy. Novel therapeutic approaches based on our expanding understanding of the mechanisms of tumor cell killing have the potential to alter this situation. Tumor suppressor gene therapy aims to restore the function of a tumor suppressor gene lost or functionally inactivated in cancer cells. One such molecule, the p53 tumor suppressor gene plays a critical role in safeguarding the integrity of the genome and preventing tumorigenesis. Our previous results not only have highlighted that apoptogenic effect of theaflavins was more pronounced in MCF-7, which contains wild type p53, than in MDA-MB-231 that has mutant p53, but also demonstrated increase in p53 level in MCF-7 cells upon theaflavin-treatment indicating the importance of p53 in theaflavin-induced breast cancer cell apoptosis. Since introduction of wild-type p53 into transformed cells has been shown to be lethal for most cancer cells in vitro, but clinical trials of p53 gene replacement have had limited success due to insufficient efficacy of current vectors and low proapoptotic activity of wt p53 as a single agent in vivo, we next attempted to study the potential therapeutic utility of combining p53 gene therapy with theaflavin-treatment in human cancer cells. To this end, we screened the cell lines expressing functional/non-functional p53 and p53-knock out cell lines to confirm the involvement of p53 in theaflavin-induced cancer cell apoptosis. Next, as the initial step to the ultimate goal of applying p53 gene therapy in cancer regression, we restored p53 expression in p53-null MDA-H0-41 cells with wild-type p53 expression plasmid. In these engineered cells theaflavins induced apoptosis dramatically although insertion of p53 alone could not increase apoptosis sufficiently when compared with p53-knock out cells. On the other hand, when wild-type p53-expressing cells were transfected with dominant-negative p53 gene, theaflavins failed to induce any apoptosis.  These results strongly reinstate our hypothesis that a combinatorial application of p53 gene therapy with theaflavin-treatment may have prospects for future investigations in the area of potential drug designing for effective cancer chemoprevention. Further work is in progress.

Cancer Immunology

To delineate the molecular mechanisms of cancer-induced immune-suppression: An approach towards immuno-editing in tumor-bearer

Amelioration of immunodepletion of cancer-bearer is another aspect of cancer management. A search for the molecular mechanisms  of tumor-induced T cell killing revealed the involvement of multiple tumor-shed immunomodulators. i) By gene-manipulation studies we show that Cox-2-dependent PGE₂ of tumor severely impairs survival signaling in CD4⁺ T cells via down-regulation of IL2Rgc expression, Jak-3/Stat-5A activation and Bcl-2/Bax ratio, thereby inducing intrinsic apoptotic pathway. Interestingly, theaflavins revert back the entire phenomena by thereby protecting CD4⁺ T cells from tumor-shed PGE₂-induced apoptosis. (ii) Tumor cells also secrete immunosuppressive cytokines, TGF-b and IL-10, while repressing Th1 cytokines, e.g., IL-2 and IFN-g, thereby inducing a general Th2 dominance dampening the Tc1 population. (iii) Tumor-shed gangliosides synergise with TNFa  to induce T cell apoptosis through receptor-dependent and -independent pathways. Interestingly, black tea and theaflavins significantly inhibited all these pro-apoptotic signalings thereby preventing T cell apoptosis in tumor-bearer. Thus, apart from its anticancer activity, this popular beverage also rejuvenates cancer immunosurveillance.