Sreeparna Chakraborty

FoxP3 Acts as a Cotranscription Factor with STAT3 in Tumor-Induced Regulatory T Cells

FoxP3, a lineage-specification factor, executes its multiple activities mostly through transcriptional regulation of target genes. We identified an interleukin-10 (IL-10)-producing FoxP3(+) T regulatory cell population that contributes to IL-10-dependent type 2 cytokine bias in breast-cancer patients. Although genetic ablation of FOXP3 inhibited IL10 transcription, genome-wide analysis ruled out its role as a transcription factor for IL10. In-depth analysis revealed that histone acetyl transterase-1, in association with FoxP3, modified the IL10 promoter epigenetically, making a space for docking STAT3-FoxP3 complexes. A predictive docking module with target-receptor specificity, along with exon-deletion and site-directed mutagenesis studies, showed that STAT3 binds through its N-terminal floppy domain to the exon 2 β sheet region of FoxP3 to form STAT3-FoxP3 complexes. Such cotranscriptional activity of FoxP3 extended to other STAT3-target genes that lack FoxP3-binding sites. These results suggest a function of FoxP3, where, failing to achieve direct promoter occupancy, FoxP3 promotes transcription in association with the locus-specific transcription factor STAT3.

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.

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.

ROS-PIASγ cross talk channelizes ATM signaling from resistance to apoptosis during chemosensitization of resistant tumors.

With the existing knowledge of ATM’s role in therapeutic resistance, the present study aimed at identifying the molecular mechanisms that influence ATM to oscillate between chemoresistance and chemosensitivity. We observed that the redox status of tumors functions as a major determinant of ATM-dependent ‘resistance-to-apoptosis’ molecular switch. At a low reactive oxygen species (ROS) condition during genotoxic insult, the ATM/sumoylated-IKKγ interaction induced NFκB activation that resisted JNK-mediated apoptosis, whereas increasing cellular ROS restored ATM/JNK apoptotic signaling. A search for the upstream missing link revealed that high ROS induces oxidation and ubiquitin-mediated degradation of PIASγ, thereby disrupting PIASγ-IKKγ cross talk, a pre-requisite for IKKγ sumoylation and subsequent NFκB activation. Interruption in the PIASγ-mediated resistance pathway channels ATM signaling toward ATM/JNK pro-death circuitry. These in vitro results also translated to sensitive and resistant tumor allograft mouse models in which low ROS-induced resistance was over-ruled in PIASγ knockout tumors, while its overexpression inhibited high ROS-dependent apoptotic cues. Cumulatively, our findings identified an unappreciated yet critical combinatorial function of cellular ROS and PIASγ in regulating ATM-mediated chemosensitization of resistant tumors. Thus, therapeutic strategies employing ROS upregulation to inhibit PIASγ during genotoxic therapy may, in future, help to eliminate the problems of NFκB-mediated tumor drug resistance.

Sulphur alters NFκB-p300 cross-talk in favour of p53-p300 to induce apoptosis in non-small cell lung carcinoma

Adverse side effects of chemotherapy during cancer treatment have shifted considerable focus towards therapies that are not only targeted but are also devoid of toxic side effects. We evaluated the antitumorigenic activity of sulphur, and delineated the molecular mechanisms underlying sulphur-induced apoptosis in non-small cell lung carcinoma (NSCLC) cells. A search for the underlying mechanism revealed that the choice between the two cellular processes, NFκBp65-mediated survival and p53-mediated apoptosis, was decided by the competition for a limited pool of transcriptional coactivator protein p300 in NSCLC cells. In contrast, sulphur inhibited otherwise upregulated survival signaling in NSCLC cells by perturbing the nuclear translocation of p65NFκB, its association with p300 histone acetylase, and subsequent transcription of Bcl-2. Under such anti-survival condition, induction of p53-p300 cross-talk enhanced the transcriptional activity of p53 and intrinsic mitochondrial death cascade. Overall, the findings of this preclinical study clearly delineated the molecular mechanism underlying the apoptogenic effect of the non-toxic homeopathic remedy, sulphur, in NSCLC cells.

Transcriptional regulation of FOXP3 requires integrated activation of both promoter and CNS regions in tumor-induced CD8 + Treg cells

T-regulatory cells are an upsurge in the tumor microenvironment and induce immune-evasion. CD4+ Treg cells are well characterized whereas the role of CD8+ Tregs in cancer has recently started to crease attention. Here, we report an augmentation CD8+FOXP3+ Tregs in breast tumor microenvironment. FOXP3, the lineage-specific transcription factor, is a dominant regulator of Treg cell development and function. FOXP3 is induced preferentially by divergent signaling in CD4+ Treg cells. But how FOXP3 is induced and maintained in tumor-CD8+ Tregs is the Cinderella of the investigation. We observed that RUNX3, a CD8+ lineage-specific transcription factor, binds at the FOXP3-promoter to induce its transcription. In addition to promoter activation, involvement of cis-elements CNS1 and CNS2 in the transcriptional regulation of FOXP3 was also evident in these cells. SMAD3 binds to CNS1 region and acts as transcription inducer, whereas GATA3 plays a temporal role in the FOXP3 transcription by differential chromatin modification in CNS regions. In CNS1 region, GATA3 acts as a repressor for FOXP3 in naïve CD8+ T cells. Whereas in CD8+ Tregs, GATA3 binds directly at CNS2 region and persuaded the maintenance of FOXP3. Therefore, the intervention of these concerted transcriptional machinery may have a therapeutic potential in immunotherapy of cancer.

Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages

Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future.

Providence of the CD25+KIR+CD127−FOXP3−CD8+ T-cell subset determines the dynamics of tumor immune surveillance

CD8+ T‐regulatory (Treg) cells are emerging as crucial components of immune system. Previous studies have reported the presence of FOXP3+CD8+ Treg cells, similar to CD4+ Tregs, in cancer patients which produce high levels of the immunosuppressive cytokines, IL10 and TGFβ. At an early stage of tumor development, we have identified a subset of FOXP3−CD8+CD25+KIR+CD127− Treg‐like cells, which are IFNγ+. However, this early‐induced CD8+CD25+CD127− T‐cell subset is certainly distinct from the IFNγ+CD8+ T‐effector cells. These CD8+CD25+CD127− T cells express other FOXP3−CD8+ Treg cell signature markers, and can selectively suppress autoreactive HLA‐E+ TFH cells as well as tumor‐induced CD4+ Treg cells. In contrast to FOXP3+CD8+ Tregs, this subset does not inhibit effector T‐cell proliferation or their functions as they are HLA‐E−. Adoptive transfer of this early‐CD8+ Treg‐like subset restrained tumor growth and inhibited CD4+ Treg generation that impedes the immune surveillance and impairs cancer immunotherapy. At the late stage of tumor development, when CD4+ Treg cells dominate the tumor‐microenvironment, CD4+ Tregs mediate the clonal deletion of these tumor‐suppressive FOXP3−IFNγ+CD8+CD25+CD127− T cells and ensure tumor immune evasion. Our findings suggest that at an early stage of the tumor, this tumor‐induced IFNγ‐producing FOXP3−CD8+CD25+CD127− T‐cell subset can potentiate immune surveillance by targeting HLA‐E‐restricted CD4+ Treg cells while leaving the effector T‐cell population unaffected. Hence, manipulating their profile can open up a new avenue in cancer immunotherapy.

Cancer-immune therapy: restoration of immune response in cancer by immune cell modulation

Immune systems play a pivotal role in recognizing cancer and induce effective immune responses for their clearance. Avoidance of immune system is one of the major hallmarks in cancer progression that successively transforms immune surveillance (tumor eradication) to immune tolerance (tumor progression). Modulation of immune cells to harness the power of effective immune responses has been long-term goals for promising strategies of cancer immune therapy. Monoclonal antibodies, immune modulators, vaccines, immune checkpoint blockers are now widely used in cancer immunotherapy. Immunotherapy also provides supportive care against high-dose cancer chemotherapy regimens. Recently immunotherapy was adopted as one of the major approaches during bone marrow transplant of hematologic malignancy. Immune-based therapeutic strategies efficiently restrict tumor evasion and have also shown efficacy against multi-drug resistant cells, one of the most crucial complications in cancer treatment. Advances in immunology and understanding the roles of immune cells in cancer microenvironment have led to numerous specific strategies to boost immune components that successively dampen cancer progression. In this review, we have described several effective immune therapy strategies that target tolerogenic immune cells to become immunogenic and restore immune surveillance in cancer. Manipulation of immune cells by novel therapeutic strategies strive to induce antitumor immune responses by expanding effective anti-tumor T cell immune responses, evoking immune activation signaling and restraining regulatory pathway that established immune-tolerance. The future of cancer immunotherapy relies on a combination of these effective strategies to harness the immune power to restrict cancer advancement.

Role of Proteases in Tumor Immune Evasion

Proteolysis or protein degradation is an important biological function which has been attributed to a major class of enzymes called proteases. In the living system, a fine-tuning between the activity of proteases and their counterparts can be seen, and disruption of this balance leads to the occurrence of various diseases including cancer. Several studies indicate that protease activity highly correlated with cancer advancement. The hallmarks of cancer like tissue evasion and metastasis, apoptosis, angiogenesis largely depend on proteolytic degradation and protease activation. Protease also contributes to avoidance of immune system, one of the emerging hallmarks in cancer progression. The immune system can recognize intracellular pathogens including cancer and elicits an effective immune response to restrict their activity and subsequently protect the host. In cancer microenvironment, immune cells are unable to recognize tumor neoantigens as well as incapable of inducing proper immune response for the clearance of tumor cells. Recent studies found that proteases not only promote tumor cell migration and metastasis it also encourages to maintain the tolerogenic tumor microenvironment by modulation of immune cell functions. Proteases are involved in several immune responses such as antigen processing and presentation, lymphocytes and neutrophil infiltration, activation of dendritic cells. The anomalies of protease actions dampen immune cell activities and establish immune tolerance. Inappropriate protease activities are responsible for inhibiting immunosurveillance and maintaining immune tolerance that ultimately leads to tumor immune evasion. Involvement of proteases in cancer suggests the use of protease inhibitors as anticancer drugs to targets not only tumor cells as well as tolerogenic immune cells to reeducate immune responses and reestablish immune surveillance. This chapter summarizes the current understanding of the interplay between proteases and the immune cells of the body and their involvement in cancer progression.