MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal

Targeting the MUC1-C oncoprotein inhibits self-renewal capacity of breast cancer cells

The capacity of breast cancer cells to form mammospheres in non-adherent serum-free culture is used as a functional characteristic of the self-renewing stem-like cell population. The present studies demonstrate that silencing expression of the MUC1-C oncoprotein inhibits growth of luminal MCF-7 and HER2-overexpressing SKBR3 breast cancer cells as mammospheres. We also show that triple-negative MDA-MB-468 breast cancer cells are dependent on MUC1-C for growth as mammospheres and tumor xenografts. Similar results were obtained when MUC1-C function was inhibited by expression of a MUC1-C(CQC→AQA) mutant. Moreover, treatment with the MUC1-C inhibitor GO-203, a cell penetrating peptide that binds to the MUC1-C cytoplasmic domain and blocks MUC1-C function, confirmed the importance of this target for self-renewal. The mechanistic basis for these findings is supported by the demonstration that MUC1-C activates NF-κB, occupies the IL-8 promoter with NF-κB, and induces IL-8 transcription. MUC1-C also induces NF-κB-dependent expression of the IL-8 receptor, CXCR1. In concert with these results, targeting MUC1-C with GO-203 suppresses IL-8/CXCR1 expression and disrupts the formation of established mammospheres. Our findings indicate that MUC1-C contributes to the self-renewal of breast cancer cells by activating the NF-κB→IL-8/CXCR1 pathway and that targeting MUC1-C represents a potential approach for the treatment of this population

MUC1-C Oncoprotein Regulates Glycolysis and Pyruvate Kinase m2 Activity in Cancer Cells

Aerobic glycolysis in cancer cells is regulated by multiple effectors that include Akt and pyruvate kinase M2 (PKM2). Mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed by human breast and other carcinomas. Here we show that transformation of rat fibroblasts by the oncogenic MUC1-C subunit is associated with Akt-mediated increases in glucose uptake and lactate production, consistent with the stimulation of glycolysis. The results also demonstrate that the MUC1-C cytoplasmic domain binds directly to PKM2 at the B- and C-domains. Interaction between the MUC1-C cytoplasmic domain Cys-3 and the PKM2 C-domain Cys-474 was found to stimulate PKM2 activity. Conversely, epidermal growth factor receptor (EGFR)-mediated phosphorylation of the MUC1-C cytoplasmic domain on Tyr-46 conferred binding to PKM2 Lys-433 and inhibited PKM2 activity. In human breast cancer cells, silencing MUC1-C was associated with decreases in glucose uptake and lactate production, confirming involvement of MUC1-C in the regulation of glycolysis. In addition, EGFR-mediated phosphorylation of MUC1-C in breast cancer cells was associated with decreases in PKM2 activity. These findings indicate that the MUC1-C subunit regulates glycolysis and that this response is conferred in part by PKM2. Thus, the overexpression of MUC1-C oncoprotein in diverse human carcinomas could be of importance to the Warburg effect of aerobic glycolysis

Potential use of liposomal diallyl sulfide in the treatment of experimental murine candidiasis

In the present study, we evaluated the potential of a liposomal formulation of the garlic oil component DAS (diallyl sulfide) in treating disseminated infection caused by the intracellular opportunistic pathogen Candida albicans in experimental mice. The PC (phosphatidylcholine) liposomal formulation of DAS was evaluated for size, ζ-potential, entrapment efficiency and release kinetics, toxicity etc. For therapeutic studies, mice were challenged with intravenous infection dosage of 107 blastospores of C. albicans followed by treatment with various doses of DAS formulations [12 and 6 mg/kg b.w. (body mass)] three times, on alternative days. The antifungal efficacy of liposomal DAS was assessed on the basis of survival of treated mice as well as the residual fungal load in vital organs like liver and spleen of mice. The results of the present study showed that treatment with DAS-bearing liposomes (12 mg/kg b.w.) resulted in the highest survival rate in animals. Liposomal DAS also significantly decreased residual fungal load in vital organs of experimental animals compared with the free form of DAS. The liposomal DAS was also found to be free of toxic manifestations as revealed by the erythrocyte lysis test and liver/kidney function tests. The results of the present study established that the antifungal activity of DAS, a poorly soluble compound, can be enhanced by the incorporation of it into liposomes. Further studies and optimizations are needed to build upon the promising findings of this study to enable the development of an effective plant-derived antifungal formulation that can provide an alternative to currently available antifungal drugs.</jats:p

Comparison of visceral leishmaniasis diagnostic antigens in African and Asian Leishmania donovani reveals extensive diversity and region-specific polymorphisms.

BACKGROUND: Visceral leishmaniasis (VL), caused by infection with Leishmania donovani complex, remains a major public health problem in endemic regions of South Asia, East Africa, and Brazil. If untreated, symptomatic VL is usually fatal. Rapid field diagnosis relies principally on demonstration of anti-Leishmania antibodies in clinically suspect cases. The rK39 immunochromatographic rapid diagnostic test (RDT) is based on rK39, encoded by a fragment of a kinesin-related gene derived from a Brazilian L. chagasi, now recognised as L. infantum, originating from Europe. Despite its reliability in South Asia, the rK39 test is reported to have lower sensitivity in East Africa. A reason for this differential response may reside in the molecular diversity of the rK39 homologous sequences among East African L. donovani strains. METHODOLOGY/PRINCIPAL FINDINGS: Coding sequences of rK39 homologues from East African L. donovani strains were amplified from genomic DNA, analysed for diversity from the rK39 sequence, and compared to South Asian sequences. East African sequences were revealed to display significant diversity from rK39. Most coding changes in the 5' half of repeats were non-conservative, with multiple substitutions involving charge changes, whereas amino acid substitutions in the 3' half of repeats were conservative. Specific polymorphisms were found between South Asian and East African strains. Diversity of HASPB1 and HASPB2 gene repeat sequences, used to flank sequences of a kinesin homologue in the synthetic antigen rK28 designed to reduce variable RDT performance, was also investigated. Non-canonical combination repeat arrangements were revealed for HASPB1 and HASPB2 gene products in strains producing unpredicted size amplicons. CONCLUSIONS/SIGNIFICANCE: We demonstrate that there is extensive kinesin genetic diversity among strains in East Africa and between East Africa and South Asia, with ample scope for influencing performance of rK39 diagnostic assays. We also show the importance of targeted comparative genomics in guiding optimisation of recombinant/synthetic diagnostic antigens