A Novel Role for Aquaporin-5 in Enhancing Microtubule Organization and Stability

Abstract Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function

Inhibition of cancer cell proliferation and apoptosis-inducing activity of fungal taxol and its precursor baccatin III purified from endophytic Fusarium solani

Background: Taxol (generic name paclitaxel), a plant-derived antineoplastic agent, used widely against breast, ovarian and lung cancer, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. The limited supply of the drug has prompted efforts to find alternative sources, such as chemical synthesis, tissue and cell cultures of the Taxus species both of which are expensive and yield low levels. Fermentation processes with microorganisms would be the methods of choice to lower the costs and increase yields. Previously we have reported that F. solani isolated from T. celebica produced taxol and its precursor baccatin III in liquid grown cultures J Biosci 33: 259-67, 2008. This study was performed to evaluate the inhibition of proliferation and induction of apoptosis of cancer cell lines by the fungal taxol and fungal baccatin III of F. solani isolated from T. celebica. Methods: Cell lines such as HeLa, HepG2, Jurkat, Ovcar3 and T47D were cultured individually and treated with fungal taxol, baccatin III with or without caspase inhibitors according to experimental requirements. Their efficacy on apoptotic induction was examined. Results: Both fungal taxol and baccatin III inhibited cell proliferation of a number of cancer cell lines with IC50 ranging from 0.005 to 0.2 mu M for fungal taxol and 2 to 5 mu M for fungal baccatin III. They also induced apoptosis in JR4-Jurkat cells with a possible involvement of anti-apoptotic Bcl2 and loss in mitochondrial membrane potential, and was unaffected by inhibitors of caspase-9,-2 or -3 but was prevented in presence of caspase-10 inhibitor. DNA fragmentation was also observed in cells treated with fungal taxol and baccatin III. Conclusions: The cytotoxic activity exhibited by fungal taxol and baccatin III involves the same mechanism, dependent on caspase-10 and membrane potential loss of mitochondria, with taxol having far greater cytotoxic potential

ARID1A-deficient bladder cancer is dependent on PI3K signaling and sensitive to EZH2 and PI3K inhibitors

Metastatic urothelial carcinoma is generally incurable with current systemic therapies. Chromatin modifiers are frequently mutated in bladder cancer, with ARID1A-inactivating mutations present in about 20% of tumors. EZH2, a histone methyltransferase, acts as an oncogene that functionally opposes ARID1A. In addition, PI3K signaling is activated in more than 20% of bladder cancers. Using a combination of in vitro and in vivo data, including patient-derived xenografts, we show that ARID1A-mutant tumors were more sensitive to EZH2 inhibition than ARID1A WT tumors. Mechanistic studies revealed that (a) ARID1A deficiency results in a dependency on PI3K/ AKT/mTOR signaling via upregulation of a noncanonical PI3K regulatory subunit, PIK3R3, and downregulation of MAPK signaling and (b) EZH2 inhibitor sensitivity is due to upregulation of PIK3IP1, a protein inhibitor of PI3K signaling. We show that PIK3IP1 inhibited PI3K signaling by inducing proteasomal degradation of PIK3R3. Furthermore, ARID1A-deficient bladder cancer was sensitive to combination therapies with EZH2 and PI3K inhibitors in a synergistic manner. Thus, our studies suggest that bladder cancers with ARID1A mutations can be treated with inhibitors of EZH2 and/or PI3K and revealed mechanistic insights into the role of noncanonical PI3K constituents in bladder cancer biology

Therapeutically actionable PAK4 is amplified, overexpressed, and involved in bladder cancer progression

Muscle-invasive bladder carcinomas (MIBCs) are aggressive genitourinary malignancies. Metastatic urothelial carcinoma of the bladder is generally incurable by current chemotherapy and leads to early mortality. Recent studies have identified molecular subtypes of MIBCs with different sensitivities to frontline therapy, suggesting tumor heterogeneity. We have performed multi-omic profiling of the kinome in bladder cancer patients with the goal of identify therapeutic targets. Our analyses revealed amplification, overexpression, and elevated kinase activity of P21 (RAC1) activated kinase 4 (PAK4) in a subset of Bladder cancer (BLCA). Using bladder cancer cells, we confirmed the role of PAK4 in BLCA cell proliferation and invasion. Furthermore, we observed that a PAK4 inhibitor was effective in curtailing growth of BLCA cells. Transcriptomic analyses identified elevated expression of another kinase, protein tyrosine kinase 6 (PTK6), upon treatment with a PAK4 inhibitor and RNA interference of PAK4. Treatment with a combination of kinase inhibitors (vandetanib and dasatinib) showed enhanced sensitivity compared with either drug alone. Thus, PAK4 may be therapeutically actionable for a subset of MIBC patients with amplified and/or overexpressed PAK4 in their tumors. Our results also indicate that combined inhibition of PAK4 and PTK6 may overcome resistance to PAK4. These observations warrant clinical investigations with selected BLCA patients