Lung cancer is one of the leading cancer malignancies with a five-year survival rate of only ~15%. We have developed a lentiviral vector mediated mouse model which allows generation of non-small cell lung cancer from less than one hundred alveolar epithelial cells, and investigated the role of IKK2 and NF-κB in lung cancer development. IKK2 depletion in tumour cells significantly attenuated tumour proliferation and significantly prolonged mouse survival. We identified Timp-1, one of the NF-κB target genes, as a key mediator for tumour growth. Activation of Erk signalling pathway and cell proliferation requires Timp-1 and its receptor CD63. Knockdown of either IKK2 or Timp-1 by shRNAs reduced tumour growth in both xenograft and lentiviral models. Our results, thus suggest the possible application of IKK2 and Timp-1 inhibitors in treating lung cancer.
PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(⌬PE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY mar , in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY mar in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY mar was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(⌬PE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(⌬PE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(⌬PE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.
In a multifaceted immunity to mycobacterial infection, induced expression of cyclooxygenase-2 (COX-2) by Mycobacterium bovis bacillus Calmette-Guerin (BCG) may act as an important influencing factor for the effective host immunity. We here demonstrate that M. bovis BCG-triggered TLR2-dependent signaling leads to COX-2 and PGE2 expression in vitro in macrophages and in vivo in mice. Further, the presence of PGE2 could be demonstrated in sera or cerebrospinal fluid of tuberculosis patients. The induced COX-2 expression in macrophages is dependent on NF-kappaB activation, which is mediated by inducible NO synthase (iNOS)/NO-dependent participation of the members of Notch1-PI-3K signaling cascades as well as iNOS-independent activation of ERK1/2 and p38 MAPKs. Inhibition of iNOS activity abrogated the M. bovis BCG ability to trigger the generation of Notch1 intracellular domain (NICD), a marker for Notch1 signaling activation, as well as activation of the PI-3K signaling cascade. On the contrary, treatment of macrophages with 3-morpholinosydnonimine, a NO donor, resulted in a rapid increase in generation of NICD, activation of PI-3K pathway, as well as the expression of COX-2. Stable expression of NICD in RAW 264.7 macrophages resulted in augmented expression of COX-2. Further, signaling perturbations suggested the involvement of the cross-talk of Notch1 with members with the PI-3K signaling cascade. These results implicate the dichotomous nature of TLR2 signaling during M. bovis BCG-triggered expression of COX-2. In this perspective, we propose the involvement of iNOS/NO as one of the obligatory, early, proximal signaling events during M. bovis BCG-induced COX-2 expression in macrophages.
Lung cancer is the most common human malignancy and leads to about one-third of all cancer-related deaths. Lung adenocarcinomas harboring KRAS mutations, in contrast to those with EGFR and EML4-ALK mutations, have not yet been successfully targeted. Here, we describe a combination therapy for treating these malignancies using two agents: a lipophilic bisphosphonate and rapamycin. This drug combination is much more effective than either agent acting alone in the KRAS G12D induced mouse lung model. Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of the KRAS and other small G-proteins critical for tumor growth and cell survival. Bisphosphonate treatment of cells initiated autophagy but was ultimately unsuccessful and led to p62 accumulation and concomitant NF-κB activation, resulting in dampened efficacy in vivo. However, we found that rapamycin, in addition to inhibiting the mTOR pathway, facilitated autophagy and prevented p62 accumulation-induced NF-κB activation and tumor cell proliferation. Overall, these results suggest that using lipophilic bisphosphonates in combination with rapamycin may provide an effective strategy for targeting lung adenocarcinomas harboring KRAS mutations.
Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-B signaling axis. Furthermore, PE_PGRS11 markedly diminished H 2 O 2 -induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.
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