Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

Deregulation of mitochondrial morphogenesis, a dynamic equilibrium between mitochondrial fusion and fission processes, is now evolving as a key metabolic event that fuels tumor growth and therapy resistance. However, fundamental knowledge underpinning how cancer cells reprogram mitochondrial morphogenesis remains incomplete. Here, we report that cystathionine ß-synthase (CBS) reprograms mitochondrial morphogenesis in ovarian cancer (OvCa) cells by selectively regulating the stability of mitofusin 2 (MFN2). Clinically, high expression of both CBS and MFN2 implicates poor overall survival of OvCa patients, and a significant association between CBS and MFN2 expression exists in individual patients in the same data set. The silencing of CBS by small interfering RNA or inhibition of its catalytic activity by a small molecule inhibitor creates oxidative stress that activates JNK. Activated JNK phosphorylates MFN2 to recruit homologous to the E6-AP carboxyl terminus' domain-containing ubiquitin E3 ligase for its degradation via the ubiquitin-proteasome system. Supplementation with hydrogen sulfide or glutathione (the catalytic products of CBS enzymatic activity), anti-oxidants, or a JNK inhibitor restores MFN2 expression. In CBS-silenced orthotopic xenograft tumor tissues, MFN2 but not MFN1 is selectively downregulated. In summary, this report reveals a role for deregulated mitochondrial morphogenesis in OvCa, suggests one of the mechanisms for this deregulation, and provides a way to correct it through modulation of the metabolic enzyme CBS.-Chakraborty, P. K., Murphy, B., Mustafi, S. B., Dey, A., Xiong, X., Rao, G., Naz, S., Zhang, M., Yang, D., Dhanasekaran, D. N., Bhattacharya, R., Mukherjee, P. Cystathionine ß-synthase regulates mitochondrial morphogenesis in ovarian cancer.

BMI1, a new target of CK2α.

BACKGROUND: The polycomb group protein, BMI1 plays important roles in chromatin modification, stem cell function, DNA damage repair and mitochondrial bioenergetics. Such diverse cellular functions of BMI1 could be, in part, due to post-translational modifications, especially phosphorylation. To date, AKT has been reported as a kinase that by site specific phosphorylation of BMI1 modulates its oncogenic functions. METHODS: Immunoprecipitation in conjunction with kinase assay and mass spectrometry was used to determine association with and site specific phosphorylation of BMI1 by CK2α. Functional implications of the BMI1/CK2α axis was examined in cancer cells utilizing siRNA and exogenous gene expression followed by biochemical and phenotypic studies. Correlations between expression of CK2α and BMI1 were determined from cell lines and formalin fixed paraffin embedded tissues representing the normal fallopian tube epithelium and high grade serous ovarian cancer samples. RESULTS: Here we report that CK2α, a nuclear serine threonine kinase, phosphorylates BMI1 at Serine 110 as determined by in-vitro/ex-vivo kinase assay and mass spectrometry. In ovarian cancer cell lines, expression of CK2α correlated with the phospho-species, as well as basal BMI1 levels. Preventing phosphorylation of BMI1 at Serine 110 significantly decreased half-life and stability of the protein. Additionally, re-expression of the phosphorylatable but not non-phosphorylatable BMI1 rescued clonal growth in endogenous BMI1 silenced cancer cells leading us to speculate that CK2α-mediated phosphorylation stabilizes BMI1 and promotes its oncogenic function. Clinically, compared to normal fallopian tube epithelial tissues, the expression of both BMI1 and CK2α were significantly higher in tumor tissues obtained from high-grade serous ovarian cancer patients. Among tumor samples, the expression of BMI1 and CK2α positively correlated (Spearman coefficient = 0.62, P = 0.0021) with each other. CONCLUSION: Taken together, our findings establish an important regulatory role of CK2α on BMI1 phosphorylation and stability and implicate the CK2α/BMI1 axis in ovarian cancer.

Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

The polycomb complex proto-oncogene BMI1 [B lymphoma Mo-MLV insertion region 1 homolog (mouse)] is essential for self-renewal of normal and cancer stem cells. BMI1-null mice show severe defects in growth, development, and survival. Although BMI1 is known to exert its effect in the nucleus via repression of 2 potent cell-cycle regulators that are encoded by the Ink4a/Arf locus, deletion of this locus only partially rescues BMI1-null phenotypes, which is indicative of alternate mechanisms of action of BMI1. Here, we show that an extranuclear pool of BMI1 localizes to inner mitochondrial membrane and directly regulates mitochondrial RNA (mtRNA) homeostasis and bioenergetics. These mitochondrial functions of BMI1 are independent of its previously described nuclear functions because a nuclear localization-defective mutant BMI1 rescued several bioenergetic defects that we observed in BMI1-depleted cells, for example, mitochondrial respiration, cytochrome c oxidase activity, and ATP production. Mechanistically, BMI1 coprecipitated with polynucleotide phosphorylase, a ribonuclease that is responsible for decay of mtRNA transcripts. Loss of BMI1 enhanced ribonuclease activity of polynucleotide phosphorylase and reduced mtRNA stability. These findings not only establish a novel extranuclear role of BMI1 in the regulation of mitochondrial bioenergetics, but also provide new mechanistic insights into the role of this proto-oncogene in stem cell differentiation, neuronal aging, and cancer.-Banerjee Mustafi, S., Aznar, N., Dwivedi, S. K. D., Chakraborty, P. K., Basak, R., Mukherjee, P., Ghosh, P., Bhattacharya, R. Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

MDR1 mediated chemoresistance: BMI1 and TIP60 in action.

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy.

G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.

Heat stress upregulates chaperone heat shock protein 70 and antioxidant manganese superoxide dismutase through reactive oxygen species (ROS), p38MAPK, and Akt.

Chinese hamster lung fibroblasts V79 cells were treated with heat stress for 4 weeks with short duration (15 min) heat shock every alternate day in culture. It was observed that Hsp 70 and the antioxidant enzyme MnSOD became overexpressed during the chronic heat stress period. Both p38 MAPK and Akt became phosphorylated by chronic heat stress exposure. Simultaneous exposure to SB203580, a potent and specific p38MAPK inhibitor drastically inhibited the phosphorylation of p38MAPK and Akt. Furthermore, exposure to SB203580 also blocked the increase in Hsp70 and MnSOD levels and the elevated SOD activity brought about by chronic heat stress. Heat shock factor 1 (HSF1) transcriptional activity and nuclear translocation of HSF1 were prominently augmented by chronic heat stress, and this amplification is markedly reduced by concomitant exposure to SB203580. Also, activations of p38MAPK and Akt and upregulations of Hsp70 and MnSOD were observed on exposure to heat shock for a single exposure of longer duration (40 min). siRNA against p38MAPK notably reduced Akt phosphorylation by single exposure to heat stress and drastically diminished the rise in Hsp70 and MnSOD levels. Similarly, siRNA against Akt also eliminated the augmentation in Hsp70 and MnSOD levels but p38MAPK levels remained unaffected. Heat stress produced reactive oxygen species (ROS) in V79 fibroblasts. N-acetyl cysteine blocked the increase in phosphorylation of p38MAPK, amplification of Hsp70, and MnSOD levels by heat stress. Therefore, we conclude that heat stress-activated p38MAPK which in turn activated Akt. Akt acted downstream of p38MAPK to increase Hsp70 and MnSOD levels.Concise summary: Thermal injury of the skin over a long period of time has been associated with development of cancerous lesions. Also, in many cancers, the cytoprotective genes Hsp70 and MnSOD have been found to be overexpressed. Therefore, we considered it important to identify the signaling elements upstream of the upregulated survival genes in heat stress. We conclude that heat stress activated p38MAPK which in turn activated Akt. Akt mediated an augmentation in Hsp70 and MnSOD levels working downstream of p38MAPK.

Inhibition of a Zn(II)-containing enzyme, alcohol dehydrogenase, by anticancer antibiotics, mithramycin and chromomycin A3.

One of the major attributes for the biological action of the aureolic acid anticancer antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) is their ability to bind bivalent cations such as Mg(II) and Zn(II) ions and form high affinity 2:1 complexes in terms of the antibiotic and the metal ion, respectively. As most of the cellular Zn(II) ion is found to be associated with proteins, we have examined the effect of MTR/CHR on the structure and function of a representative structurally well characterized Zn(II) metalloenzyme, alcohol dehydrogenase (ADH) from yeast. MTR and CHR inhibit enzyme activity of ADH with inhibitory constants of micromolar order. Results from size-exclusion column chromatography, dynamic light scattering, and isothermal titration calorimetry have suggested that the mechanism of inhibition of the metalloenzyme by the antibiotics is due to the antibiotic-induced disruption of the enzyme quaternary structure. The nature of the enzyme inhibition, the binding stoichiometry of two antibiotics per monomer, and comparable dissociation constants for the antibiotic and free (or substrate-bound) ADH imply that the association occurs as a consequence of the binding of the antibiotics to Zn(II) ion present at the structural center. Confocal microscopy shows the colocalization of the antibiotic and the metalloenzyme in HepG2 cells, thereby supporting the proposition of physical association between the antibiotic(s) and the enzyme inside the cell.

Pro-survival effects of repetitive low-grade oxidative stress are inhibited by simultaneous exposure to Resveratrol.

V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30 microM H(2)O(2) for 4 weeks. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing agent such as ultraviolet radiation (UVR). Concurrent treatment with Resveratrol completely restored the normal apoptotic response after UVR. p38MAPK became dually phosphorylated during the stress period. Akt also became phosphorylated on Ser(473) in cells subjected to repetitive oxidative stress. In these cells, NFkappaB p65 became phosphorylated and appreciable nuclear localization of p65 was observed. NFkappaB transcriptional activity also became augmented during repetitive stress. Treatment of the repetitively stressed cells concurrently with Resveratrol or SB203580, a p38MAPK inhibitor, robustly blocked activation of p38MAPK, NFkappaB transcriptional activity, phosphorylation and nuclear localization of p65, and Akt phosphorylation. Pre-exposure to short interfering RNA (si RNA) to p38MAPK, resulted in a blockage of the Akt and NFkappaB p65 phosphorylation. However, inhibition of Akt activity through PI3 kinase inhibitor LY294002 did not result in obstruction of p38MAPK phosphorylation by H(2)O(2). Also, Resveratrol was effective as an antioxidant in counteracting a rise in reactive oxygen species (ROS) and p38MAPK activation by H(2)O(2) was completely blocked by antioxidant N-acetyl cysteine (NAC). We conclude that Resveratrol acts as an antioxidant and completely reverses the anti-apoptotic effects of repetitive stress by blocking oxidative stress-induced p38MAPK activation which is the key regulatory step for the activation of down-stream survival elements Akt and NFkappaB.

Activation of p38MAPK by repetitive low-grade oxidative stress leads to pro-survival effects.

V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30 microM H2O2 for 4 weeks. Within the first week of treatment p38MAPK became dually phosphorylated and became increasingly phosphorylated during the 4-week stress period. Akt also became phosphorylated on Ser473 and Thr308 after the second week of treatment and remained phosphorylated throughout the study. NFkappaB p65 and IkappaB kinase (IKK) became phosphorylated and NFkappaB transcriptional activity became augmented during repetitive stress. Treatment of the cells concurrently with SB203580, a specific p38MAPK inhibitor, robustly blocked activation of NFkappaB transcriptional activity, phosphorylation of p65, and IKK but only partially blocked Akt phosphorylation. Similar simultaneous treatment with PI-3 kinase inhibitor LY294002 prominently blocked Akt phosphorylation. Pre-exposure to short interfering RNA (si RNA ) to p38MAPK resulted in a complete blockage of the NFkappaB p65 and IKK phosphorylations as well as the anti-apoptotic influence induced by a single low dose of H2O2 but produced a partial obstruction of Akt phosphorylation. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing agents such as ultraviolet radiation (UVR) and mM H2O2. Concurrent treatment with SB203580 almost completely restored the normal apoptotic response such as DNA fragmentation after UVR and mM H2O2. LY294002, a PI-3 kinase inhibitor and SN-50, an inhibitor of NFkappaB, produced partial restorations of the apoptotic response. We conclude that activation of p38MAPK by repetitive oxidative stress is the key event which through its command over down-stream survival elements such as Akt and NFkappaB controls the anti-apoptotic environment of the repetitively H2O2-stressed cells.

Tea polyphenol epigallocatechin 3-gallate impedes the anti-apoptotic effects of low-grade repetitive stress through inhibition of Akt and NFkappaB survival pathways.

V79 Chinese Hamster lung fibroblasts were subjected to repetitive low-grade stress through multiple exposures to 30 microM H2O2 in culture for 4 weeks. Akt/protein kinase B became phosphorylated at serine473 and threonine308 during this period of repetitive stress. Concurrent exposure of the cells to LY294002 (5 microM), a phosphoinositide-3 kinase inhibitor or 4.5 microM epigallocatechin 3-gallate (EGCG), a tea polyphenol almost completely blocked Akt activation by repetitive stress. Phosphorylation of I kappa B kinase (IKK) and transcriptional activity driven by nuclear factor kappa B (NFkappaB) were significantly enhanced by repetitive oxidative stress. These increases were largely abolished by simultaneous exposure to EGCG. The repetitively stressed cells demonstrated a significant resistance to apoptosis by subsequent acute stress in the form of ultraviolet radiation at 5 J/m2 or H2O2 (7.5 mM). The resistance to apoptosis conferred by repetitive stress was drastically reduced (>80%) by constant exposure to EGCG during the stress period while the presence of LY294002 or the NFkappaB inhibitor SN50 brought about a relatively moderate effect (about 50-65%). Our data indicate that activation of Akt and NFkappaB pro-survival pathways by repetitive low-grade stress results in a strong inhibition of the normal apoptotic response after subsequent acute stress. The tea polyphenol EGCG impedes the activation of both Akt and NFkappaB by repetitive stress and as a result preserves the normal apoptotic response during subsequent acute stress.

p38 mitogen-activated protein kinase (p38MAPK) upregulates catalase levels in response to low dose H2O2 treatment through enhancement of mRNA stability.

V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 microM) H2O2 in culture for 4 weeks. Catalase activity, protein levels and mRNA levels increased markedly (5-6-fold) during this time and these augmentations were inhibited by the simultaneous presence of SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK). p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Short interfering RNA that induced effective silencing of p38MAPK, was used to silence p38MAPK in V79 fibroblasts. Silencing of p38MAPK drastically hindered the elevation in catalase (protein and mRNA) levels observed after a single low dose (50 microM) of H2O. The rise in catalase mRNA levels induced by low concentration (single and multiple dose) H2O2 treatment was established to be unconnected with transcriptional upregulation but was brought forth primarily by an enhancement in catalase mRNA stability through the action of p38MAPK. Therefore, our data strongly indicate that activation of p38MAPK is a key controlling step in the upregulation of catalase levels by low dose H2O2 treatment.