SifA SUMOylation governs Salmonella Typhimurium intracellular survival via modulation of lysosomal function.

One of the mechanisms shaping the pathophysiology during the infection of enteric pathogen Salmonella Typhimurium is host PTM machinery utilization by the pathogen encoded effectors. Salmonella Typhimurium (S. Tm) during infection in host cells thrives in a vacuolated compartment, Salmonella containing vacuole (SCV), which sequentially acquires host endosomal and lysosomal markers. Long tubular structures, called as Salmonella induced filaments (SIFs), are further generated by S. Tm, which are known to be required for SCV's nutrient acquisition, membrane maintenance and stability. A tightly coordinated interaction involving prominent effector SifA and various host adapters PLEKHM1, PLEKHM2 and Rab GTPases govern SCV integrity and SIF formation. Here, we report for the first time that the functional regulation of SifA is modulated by PTM SUMOylation at its 11th lysine. S. Tm expressing SUMOylation deficient lysine 11 mutants of SifA (SifAK11R) is defective in intracellular proliferation due to compromised SIF formation and enhanced lysosomal acidification. Furthermore, murine competitive index experiments reveal defective in vivo proliferation and weakened virulence of SifAK11R mutant. Concisely, our data reveal that SifAK11R mutant nearly behaves like a SifA knockout strain which impacts Rab9-MPR mediated lysosomal acidification pathway, the outcome of which culminates in reduced bacterial load in in vitro and in vivo infection model systems. Our results bring forth a novel pathogen-host crosstalk mechanism where the SUMOylation of effector SifA regulated S. Tm intracellular survival.

Transcriptional regulation of VEGFA expression in T-regulatory cells from breast cancer patients.

The initiation of new blood vessel formation (neo-angiogenesis) is one of the primary requirements for the establishment of tumor. As the tumor grows beyond a certain size, a hypoxic-condition arises in the inner core of tumor, triggering the release of chemokines, which attract T-regulatory (Treg) cells in the tumor-site. The presence of FOXP3, a lineage-specific transcription factor, expressing Treg cells in various types of tumor implements immunosuppressive and tumor-promoting strategies. One such strategy is the invitation of endothelial cells for neo-vascularization in the tumor site. Here we report that as the disease progresses, Treg cells from breast cancer patients are capable of secreting high-amount of VEGFA. The VEGFA promoter lacks Treg-specific transcription factor FOXP3 binding site. FOXP3 in association with locus-specific transcription factor STAT3 binds to VEGFA promoter to induce its transcription in Treg cells obtained from breast cancer patients. Treg cell-secreted VEGFA induces neo-angiogenesis from endothelial cells under in-vitro conditions. Targeting Tregs in mice with breast tumor reduces tumor growth as well as the level of neo-angiogenesis in the tumor tissue.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

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 TGFß. 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.

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.

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.

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.

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.

MEK inhibition prevents tumour-shed transforming growth factor-ß-induced T-regulatory cell augmentation in tumour milieu.

Tumour progression is associated with immune-suppressive conditions that facilitate the escape of tumour cells from the regimen of immune cells, subsequently paralysing the host defence mechanisms. Induction of CD4(+)  CD25(+)  FoxP3(+) T regulatory (Treg) cells has been implicated in the tumour immune escape mechanism, although the novel anti-cancer treatment strategies targeting Treg cells remain unknown. The focus of this study is to define the interaction between tumour and immune system, i.e. how immune tolerance starts and gradually leads to the induction of adaptive Treg cells in the tumour microenvironment. Our study identified hyperactivated mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) -signalling as a potential target for reversing Treg cell augmentation in breast cancer patients. In more mechanistic detail, pharmacological inhibitors of MEK/ERK signalling inhibited transforming growth factor-ß (TGF-ß) production in tumour cells that essentially blocked TGF-ß-SMAD3/SMAD4-mediated induction of CD25/interleukin-2 receptor α on CD4(+) T-cell surface. As a result high-affinity binding of interleukin-2 on those cells was prohibited, causing lack of Janus kinase 1 (JAK1)/JAK3-mediated signal transducer and activator of transcription 3 (STAT3)/STAT5 activation required for FoxP3 expression. Finally, for a more radical approach towards a safe MEK inhibitor, we validate the potential of multi-kinase inhibitor curcumin, especially the nano-curcumin made out of pure curcumin with greater bioavailability; in repealing tumour-shed TGF-ß-induced Treg cell augmentation.