Upregulation Of Mcl-1 Research Articles

Abstract C205: Cytotoxicity and target modulation in pediatric solid tumors by the proteasome inhibitor carfilzomib.

Abstract Rationale: Currently, even with escalating multimodal treatment regimens, most children with recurrent metastatic solid tumors endure unacceptably high mortality rates. Hence, there is an urgent need to identify novel targets and new therapeutic approaches. Recent studies have shown that proteasome inhibition leads to effective tumor killing in cells that have acquired treatment resistance and metastatic properties. Carfilzomib (CFZ) is a selective and potent proteasome inhibitor that binds and inhibits the 20S proteasome resulting in the accumulation of polyubiquinated proteins and consequently, cycle arrest, growth inhibition and apoptosis. Clinical trials in adult myeloma have shown activity and a high tolerability of CFZ. However, data with respect to the potential of this agent for refractory pediatric solid tumors are not yet available. Methods: Cells from a panel of lines including neuroblastoma (n=6), Ewings sarcoma (n=2), osteosarcoma (n=2), rhabdomyosarcoma (n=4) and ATRT (n=2), were treated with increasing concentrations of CFZ and cell growth inhibition was quantified by Alamar blue assay. Drug pulsing experiments were carried out by adding new drug preparations at defined time intervals. Target modulation and apoptosis analyses were done by Western blotting. Drug combination studies were interpreted using the Chou and Talalay method. Results and Discussion: CFZ showed effective cytotoxicity against all cell lines tested (mean IC50 = 7nM, range = 1-20nM) and activity in a fluorophore tagged cell based proteasome assay. More detailed target modulation studies in NB cells showed a dose dependent initial up-regulation of MCL-1 that subsequently decreased in a manner corresponding with PARP cleavage. Up-regulation of BCL-2 was also noted but required the exposure to higher drug concentrations. This indicated the initial stabilization or activation of survival pathways in response to drug treatment. Drug scheduling showed that the minimum exposure of 4 to 8 hours /day is needed for effective cumulative killing coinciding with MCL-1 regulation. Drug combination studies identified the ability of CFZ to synergistically enhance the activity of a number of chemotherapeutic agents, including etoposide, vincristine, mefloquine and the BCL-2 antagonist ABT-263. However, the extent of synergy differed greatly between cell types indicating the potentially variable relationship of proteasome functions to distinct oncogenic pathways present in different cells. Our studies provide initial in vitro data on the potential of CFZ to treat pediatric solid tumors and support further investigations in to the components of drug scheduling, biological correlates and drug combinations for future early phase clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C205. Citation Format: Yibing Ruan, David Liu, Aarthi Jayanthan, Tony Truong, Jessica Boklan, Aru Narendran. Cytotoxicity and target modulation in pediatric solid tumors by the proteasome inhibitor carfilzomib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C205.

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1.

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.

Abstract B071: Targeting the antiapoptotic protein Mcl-1 in luminal B breast cancers

Abstract In an effort to improve therapeutic tumor cell killing in luminal B breast cancers, we are investigating the combination of endocrine therapies with targeted inhibition of pro-survival proteins of the Bcl2 family, including Bcl2, Bcl-xL, and Mcl-1. Bcl2-family proteins are often overexpressed in breast cancers and are associated with tumor progression, therapeutic resistance, and poor patient survival. These proteins function by sequestering pro-apoptotic BH3-only proteins (BIM, BID, Noxa and Puma), thus inhibiting caspase-dependent apoptosis. We found Mcl1 gene amplification in 15/133 luminal B breast tumors (11%) curated through the Cancer Genome Atlas (TCGA), and Bcl2l1 (encoding Bcl-xL) gene amplification in 1/133 luminal B breast tumors, although Bcl2l1 mRNA was upregulated in 19/133 samples. Bcl2 was not amplified or overexpressed in TCGA-curated luminal B breast tumors. The BH3 mimetic ABT-263, which specifically binds Bcl2 and Bcl-xL, increased tumor cell killing in 3 out of 4 estrogen-deprived luminal B human breast cancer cell lines. However, ZR75-1 cells, which are Mcl1 gene amplified, were resistant to ABT-263. Further, Mcl-1 protein upregulation was induced in HCC-1428 and T47D cells treated with ABT-263, suggesting that cells may use Mcl-1 upregulation to partially mitigate the effects of Bcl-xL targeting with ABT-263. Mcl-1 knockdown using an shRNA approach, decreased cell growth and increased caspase-dependent apoptosis, and increased sensitivity to ABT-263 in 4 of the 4 cell lines tested, including ZR75-1 cells. We used long-term estrogen withdrawal (LTED) to model the sustained response of breast cancer cells to aromatase inhibitors (e.g., exemestane). As compared to parental breast cancer cell lines under estrogen withdraw conditions, LTED populations drastically increased protein levels of Mcl-1. Future experiments will be aimed at assessing the impact of Mcl-1 depletion on ABT-263-treated LTED populations. We predict that combined inhibition of Bcl-2, Bcl-xL and Mcl-1 will improve response of cells to estrogen deprivation. In summary, these preliminary studies suggest that BH3 mimetics targeting Bcl-2 family proteins may be therapeutically efficacious in endocrine-refractory luminal B breast tumors, and support future endeavors to generate BH3-mimetics specifically targeting Mcl-1. Citation Format: Michelle M. Williams, Steve Fesik, Donna Hicks, Rebecca S. Cook. Targeting the antiapoptotic protein Mcl-1 in luminal B breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B071.

Overexpression of p-Stat3 and Mcl-1, and their correlation with differentiation and apoptotic resistance in esophageal squamous cell carcinoma

To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed. Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012). In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL-15 is an essential survival factor for CD8αα(+) intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL-15-induced survival signals in primary CD8αα(+) iIELs remains elusive. Although Bcl-2 level in CD8αα(+) iIELs positively correlates with IL-15Rα expression in the intestinal epithelial cells, overexpression of Bcl-2 only moderately restores CD8αα(+) γδ iIELs in Il15(-/-) mice. Here, we found that IL-15 promptly activated a Jak3-Jak1-PI3K-Akt pathway that led to the upregulation of Bcl-2 and Mcl-1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl-2 but also resulted in the phosphorylation of extra-long Bim at Ser(65) . The latter event facilitated the dissociation of Bim from Bcl-2 without affecting Bim abundance in IL-15-treated CD8αα(+) iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl-2 or removal of Bim from CD8αα(+) iIELs promoted their survival in Il15ra(-/-) mice. Taken together, IL-15 promotes CD8αα(+) iIEL survival by both increasing Bcl-2 levels and dissociating Bim from Bcl-2 through activation of a Jak3-Jak1-PI3K-Akt-ERK1/2 pathway, which differs from a previously reported IL-15-induced survival signal.

Epidermal Growth Factor Receptor (EGFR) Signaling Promotes Proliferation and Survival in Osteoprogenitors by Increasing Early Growth Response 2 (EGR2) Expression

Maintaining bone architecture requires continuous generation of osteoblasts from osteoprogenitor pools. Our previous study of mice with epidermal growth factor receptor (EGFR) specifically inactivated in osteoblast lineage cells revealed that EGFR stimulates bone formation by expanding the population of mesenchymal progenitors. EGFR ligands are potent regulators for the osteoprogenitor pool, but the underlying mechanisms are largely unknown. Here we demonstrate that activation of EGFR increases the number of osteoprogenitors by promoting cell proliferation and suppressing either serum depletion-induced or TNFα-induced apoptosis mainly through the MAPK/ERK pathway. Mouse calvarial organ culture revealed that EGF elevated the number of proliferative cells and decreased the number of apoptotic cells, which led to increased osteoblasts. Microarray analysis of MC3T3 cells, an osteoprogenitor cell line, revealed that EGFR signaling stimulates the expression of MCL1, an antiapoptotic protein, and a family of EGR transcription factors (EGR1, -2, and -3). The up-regulation of MCL1 and EGR2 by EGF was further confirmed in osteoprogenitors close to the calvarial bone surface. Overexpression of NAB2, a co-repressor for EGRs, attenuated the EGF-induced increase in osteoprogenitor number. Interestingly, knocking down the expression of EGR2, but not EGR1 or -3, resulted in a similar effect. Using inhibitor, adenovirus overexpression, and siRNA approaches, we demonstrate that EGFR signaling activates the MAPK/ERK pathway to stimulate the expression of EGR2, which in turn leads to cell growth and MCL1-mediated cell survival. Taken together, our data clearly demonstrate that EGFR-induced EGR2 expression is critical for osteoprogenitor maintenance and new bone formation.

Abstract 3290: Modulation of AKT, ERK and Mcl-1 by apigenin sensitizes colon cancer cells to anti-tumor activities of ABT263.

Abstract Apigenin is an edible plant-derived flavonoid showing anti-tumor activities in vitro and in vivo. Pretreatment with apigenin resulted in cell growth arrest and/or apoptosis in various types of tumors via modulating multiple cell signaling pathways. In the present study, we evaluated interactions between apigenin and ABT263 in multiple colon cancer cell lines. We found that apigenin enhanced ABT263-induced apoptosis in colon cancer cells in a time- and dose-dependent manner. ABT263 alone only resulted in limited cell killing in association with upregulation of Mcl-1, a potential mechanism for acquired ABT263 resistance. Apigenin antagonized ABT263-induced Mcl-1 expression and dramatically enhanced ABT263-induced cell death. Apigenin also suppressed AKT and ERK activation. Inactivation of either AKT or ERK pathway by lentivirus-transduced shRNA or specific small molecule inhibitors synergized with ABT263 to induce cell death, reflecting that AKT and ERK pathways are apigenin targets. Moreover, the synergism between ABT263 and apigenin was associated with the upregulation of Bim and subsequent activation of Bax. Xenograft studies with HCT116 cells in SCID mice showed that the combination treatment with apigenin and ABT263 inhibited tumor growth up to 70% without obvious adverse effects, while either agent alone showed only about 30% inhibition. Collectively, our findings demonstrate a novel and effective strategy to treat colon cancer by combination of ABT263 and apigenin. The combined effect is mediated by apigenin downregulation of Mcl-1, Akt and ERK activities. Citation Format: Huanjie Shao, Kai Jing, Esraa Mahmoud, Xianjun Fang, Chunrong Yu. Modulation of AKT, ERK and Mcl-1 by apigenin sensitizes colon cancer cells to anti-tumor activities of ABT263. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3290. doi:10.1158/1538-7445.AM2013-3290

Abstract 4412: S1 and gossypol induce NOXA through increased reactive oxygen species or cytoplasmic calcium and sensitize to ABT-737.

Abstract The upregulation of anti-apoptotic BCL2 family members enhances the survival of cancer cells, such as in chronic lymphocytic leukemia (CLL). A class of inhibitors (termed BH3 mimetics) has been developed to inhibit BCL2 proteins and selectively kill cancer cells. These inhibitors function by occupying the groove through which BCL2 proteins interact with their binding partners. ABT-737 is a high affinity inhibitor of BCL2 and BCLXL, and navitoclax, which has a similar binding profile, has shown efficacy in clinical trials. However, resistance occurs when the cancer cells rely on additional BCL2 members, such as MCL1 or BFL1, which are not inhibited by ABT-737. Using leukemia cell lines, we compared several BH3 mimetics reported to inhibit multiple BCL2-family proteins (including MCL1 and BFL1), with the goal of enhancing the efficacy of ABT-737. We found that none of these compounds functioned as true BH3 mimetics. Instead, the compounds induced ER stress and upregulated the pro-apoptotic protein NOXA. Furthermore, two of these compounds, S1 and gossypol, sensitized leukemia cells to ABT-737 in a NOXA-dependent manner. NOXA induced by S1 or gossypol is dependent on increased reactive oxygen species or increased cytoplasmic calcium, respectively. We also tested these drug combinations in CLL cells ex vivo. S1 and gossypol combined with ABT-737 to selectively kill CLL cells but not normal lymphocytes. Interaction between CLL cells and stroma in patients promotes resistance to BCL2 inhibitors through the upregulation of MCL1 and BFL1. Co-incubation of CLL cells ex vivo with CD40 ligand-expressing stroma mimics the resistance to BCL2 inhibitors seen in patients. Importantly, this resistance was overcome when S1 or gossypol was combined with ABT-737. Our results show two novel mechanisms for upregulating NOXA and sensitizing leukemia cells to ABT-737. These combinations also kill CLL cells exposed to stroma, and may therefore target residual CLL cells in vivo, thus improving clinical outcome. Citation Format: Ryan S. Soderquist, Darcy J. P. Bates, Alan Eastman. S1 and gossypol induce NOXA through increased reactive oxygen species or cytoplasmic calcium and sensitize to ABT-737. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4412. doi:10.1158/1538-7445.AM2013-4412

Abstract 1727: Evidence that E2F1-mediated upregulation of Bim, a proapoptotic BH3-only protein, functions as a prosurvival molecule in cancer.

Abstract Proapoptotic BH3-only protein Bim plays an important role in Bax/Bak-mediated cytochrome c release and apoptosis. Here we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally upregulated. We have identified eight endogenous E2F1-binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1-dependent as mutation of E2F1-binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton-associated fractions. Bim-silencing or microinjection of anti-Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed upregulation of prosurvival proteins Bcl-xL and Mcl-1, which sequester Bim in cancer cells. In addition, phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl-xL/Mcl-1 block its proapoptotic functions. The prosurvival functions associated with multiple proapoptotic proteins raise the possibility that anticancer agents that only harness proapoptotic function of Bim or other BH3-only proteins may not be an efficient approach for the permeabilization of mitochondria. Our findings demonstrate that in order to induce efficient apoptosis, inhibition of prosurvival function of Bim may provide a new approach for efficient cancer therapy. Citation Format: Neelu Yadav, Raghu Gogada, Junwei Liu, Shaohua Tang, Dianmu Zhang, C Marcelo Aldaz, Dean Tang, Dhyan Chandra. Evidence that E2F1-mediated upregulation of Bim, a proapoptotic BH3-only protein, functions as a prosurvival molecule in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1727. doi:10.1158/1538-7445.AM2013-1727

Abstract 601: Antioxidant inhibition of GSK-3β stabilizes anti-apoptotic factor Mcl-1 leading to decreased apoptosis and increased cell survival.

Abstract Antioxidant-mediated stabilization of Nrf2 is known to up-regulate anti-apoptotic factors Bcl-2 and Bcl-xL that leads to reduced apoptosis and increased cell survival. In this report we investigated and found that anti-apoptotic factor Mcl-1 is coordinately induced with Bcl-2 and Bcl-xL factors in response to antioxidants. However, unlike Bcl-2 and Bcl-xL, Nrf2 did not control antioxidant-mediated up-regulation of Mcl-1. Further studies revealed that ERK1/2 phosphorylation of Mcl-1T163 primes Mcl-1 for phosphorylation by GSK3β at position S159. This modification degrades Mcl-1 and contributes to the maintenance of physiological level of Mcl-1 and cellular apoptosis. Exposure of Hep-G2 cells to antioxidant tert-butyl hydroquinone (t-BHQ) for 4-16h led to time dependent Akt-mediated inactivation of GSK3β, decreased Mcl-1S159 phosphorylation resulting in stabilization of Mcl-1 protein. Antioxidant-mediated increased Mcl-1 heterodimerized with Bim and Bax and down regulated cellular apoptosis. The contribution of antioxidant increased Mcl-1 in reduced apoptosis was further confirmed by siRNA-mediated inhibition of Mcl-1 that increased susceptibility to etoposide-mediated cell death and reduced cell survival through the activation of caspases 3/7. These results provided evidence that antioxidant control of GSK-3β led to increase in Mcl-1 that contributed to decreased apoptosis and increased cell survival. The results collectively suggest that antioxidant stabilized Nrf2 and GSK3β both through distinct mechanisms lead to increased anti-apoptotic factors Bcl-2, Bcl-xL and Mcl-1 that reduces apoptosis and promotes cell survival. Citation Format: Suryakant K. Niture, Anil K. Jaiswal. Antioxidant inhibition of GSK-3β stabilizes anti-apoptotic factor Mcl-1 leading to decreased apoptosis and increased cell survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 601. doi:10.1158/1538-7445.AM2013-601

Abstract 3072: Characterization of TCL1-Tg:p53-/- mice that resemble human chronic lymphocytic leukemia with 17p-deletion: alterations in p53→Mir30→EZH2 axis.

Abstract Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United States and Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. We have recently generated mice with Eμ-Tcl1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. In the present study, we further demonstrated that the p53 deficiency in the Tcl1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family in leukemia lymphocytes from the Eμ-Tcl1-Tg:p53-/- mice. Consistently, primary leukemia cells from CLL patients with 17p deletion also showed a decrease in the miR-30d expression. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eμ-Tcl1-Tg:p53-/- mice relative to Eμ-Tcl1-Tg:p53+/+ mice. Exposure of leukemia cells isolated from Eμ-Tcl1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study reveals that the p53-miR30-EZH2 axis may play an important role in CLL pathogenesis, and EZH2 may potentially be a target for CLL treatment. Citation Format: Jinyun Liu, Marcia A. Ogasawara, Gang Chen, Helene Pelicano, Peng Huang. Characterization of TCL1-Tg:p53-/- mice that resemble human chronic lymphocytic leukemia with 17p-deletion: alterations in p53→Mir30→EZH2 axis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2013-3072

Evidence that E2F1‐mediated upregulation of Bim, a proapoptotic BH3‐only protein, functions as a prosurvival molecule in cancer

Proapoptotic BH3‐only protein Bim plays an important role in Bax/Bak‐mediated cytochrome c release and apoptosis. Here we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally upregulated. We have identified eight endogenous E2F1‐binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1‐dependent as mutation of E2F1‐binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton‐associated fractions. Bim‐silencing or microinjection of anti‐Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed upregulation of prosurvival proteins Bcl‐xL and Mcl‐1, which sequester Bim in cancer cells. In addition, phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl‐xL/Mcl‐1 block its proapoptotic functions.

Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

Ursolic acid (UA) is a pentacyclic triterpenoid with promising cancer chemopreventive properties. A better understanding of the mechanisms underlying anticancer activity of UA is needed for further development as a clinically useful chemopreventive agent. Here, we found that both endoplasmic reticulum (ER) stress and autophagy were induced by UA in MCF-7 human breast cancer cells. Surprisingly, ER stress was identified as an effect rather than a cause of UA-induced autophagy. Autophagy-dependent ER stress protected the cells from UA-induced apoptosis through EIF2AK3-mediated upregulation of MCL1. Activation of MAPK1/3 but not inhibition of MTOR pathway contributed to UA-induced cytoprotective autophagy in MCF-7 cells. Our findings uncovered a novel cellular mechanism involved in the anticancer activity of UA, and also provided a useful model to study biological significance and mechanisms of autophagy-mediated ER stress.

Bif-1 haploinsufficiency promotes chromosomal instability and accelerates Myc-driven lymphomagenesis via suppression of mitophagy

AbstractMalignant transformation by oncogenes requires additional genetic/epigenetic changes to overcome enhanced susceptibility to apoptosis. In the present study, we report that Bif-1 (Sh3glb1), a gene encoding a membrane curvature–driving endophilin protein, is a haploinsufficient tumor suppressor that plays a key role in the prevention of chromosomal instability and suppresses the acquisition of apoptosis resistance during Myc-driven lymphomagenesis. Although a large portion of Bif-1–deficient mice harboring an Eμ-Myc transgene displayed embryonic lethality, allelic loss of Bif-1 dramatically accelerated the onset of Myc-induced lymphoma. At the premalignant stage, hemizygous deletion of Bif-1 resulted in an increase in mitochondrial mass, accumulation of DNA damage, and up-regulation of the antiapoptotic protein Mcl-1. Consistently, allelic loss of Bif-1 suppressed the activation of caspase-3 in Myc-induced lymphoma cells. Moreover, we found that Bif-1 is indispensable for the autophagy-dependent clearance of damaged mitochondria (mitophagy), because loss of Bif-1 resulted in the accumulation of endoplasmic reticulum–associated immature autophagosomes and suppressed the maturation of autophagosomes. The results of the present study indicate that Bif-1 haploinsufficiency attenuates mitophagy and results in the promotion of chromosomal instability, which enables tumor cells to efficiently bypass the oncogenic/metabolic pressures for apoptosis.

Bim, a Proapoptotic Protein, Up-regulated via Transcription Factor E2F1-dependent Mechanism, Functions as a Prosurvival Molecule in Cancer

Proapoptotic Bcl-2 homology 3-only protein Bim plays an important role in Bax/Bak-mediated cytochrome c release and apoptosis. Here, we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally up-regulated. We have identified eight endogenous E2F1-binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1-dependent as mutation of the E2F1-binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton-associated fractions. Bim silencing or microinjection of anti-Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed up-regulation of prosurvival proteins Bcl-xL and Mcl-1, which sequester Bim in cancer cells. In addition, a phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl-xL/Mcl-1 block its proapoptotic functions.

Apoptotic Machinery Diversity in Multiple Myeloma Molecular Subtypes

Multiple myeloma (MM) is a plasma-cell (PC) malignancy that is heterogeneous in its clinical presentation and prognosis. Monoclonal gammopathy of undetermined significance (MGUS) consistently preceded development of MM. The presence of primary IgH translocations and the universal overexpression of cyclin D genes led to a molecular classification of MM patients into different disease subtypes. Since Bcl-2 family proteins determine cell fate, we analyzed a publicly available Affymetrix gene expression of 44 MGUS and 414 newly diagnosed MM patients to investigate (1) the global change of Bcl-2 family members in MM versus MGUS (2) whether the four major subtypes defined as hyperdiploid, CyclinD1, MAF, and MMSET, display specific apoptotic machineries. We showed that among the main anti-apoptotic members (Bcl-2, Bcl-xL, and Mcl-1), Mcl-1 up-regulation discriminated MM from MGUS, in agreement with the prominent role of Mcl-1 in PC differentiation. Surprisingly, the expression of multi-domain pro-apoptotic Bak and Bax were increased during the progression of MGUS to MM. The combined profile of Bcl-2, Bcl-xL, and Mcl-1 was sufficient to distinguish MM molecular groups. While specific pro-apoptotic members expression was observed for each MM subtypes, CyclinD1 subgroup, was identified as a particular entity characterized by a low expression of BH3-only (Puma, Bik, and Bad) and multi-domain pro-apoptotic members (Bax and Bak). Our analysis supports the notion that MM heterogeneity is extended to the differential expression of the Bcl-2 family content in each MM subgroup. The influence of Bcl-2 family profile in the survival of the different patient groups will be further discussed to establish the potential consequences for therapeutic interventions. Finally, the use of distinct pro-survival members in the different steps of immune responses to antigen raises also the question of whether the different Bcl-2 anti-apoptotic profile could reflect a different origin of MM cells.

Regulation of Mcl-1 by SRSF1 and SRSF5 in Cancer Cells

Up-regulation of the apoptosis-regulatory gene Mcl-1 (myeloid cell leukemia-1) occurs in different cancer types and is linked with drug resistance to cancer therapies. It is well known that Mcl-1 pre-mRNA undergoes alternative splicing events to produce two functionally distinct proteins, Mcl-1S (pro-apoptotic) and Mcl-lL (anti-apoptotic); the latter isoform is predominant in different cancers including breast and ovarian cancer cells. In the present study we report that the RNA-binding protein (RBP) and proto-oncogene SRSF1 (serine and arginine-rich splicing factor 1) influences splicing of Mcl-1 in both MCF-7 and MDA-MB-231 breast cancer cells and JAR choriocarcinoma cells; we also show for the first time that another RBP SRSF5 affects splicing of Mcl-1 in the MCF-7 cells. Moreover, we report that SRSF1 is involved in other aspects of Mcl-1 regulation with knockdown of SRSF1, by RNAi, resulting in a significant decrease in Mcl-1 protein levels in MCF-7 cells but an increase in JAR cells, respectively, by potentially affecting protein stability and translation of Mcl-l. The key findings from this study highlight the importance of the cellular context of different cancer cells for the function of multifunctional RBPs like SRSF1 and have implications for therapeutic approaches employed to target Mcl-1.

Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis

BackgroundAscites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.MethodsUsing human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.ResultsIn the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.ConclusionsThese results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

Bortezomib/proteasome inhibitor triggers both apoptosis and autophagy-dependent pathways in melanoma cells

Generally, both endoplasmic reticulum (ER) stress and mitochondrial dysregulation are a potential therapeutic target of anticancer agents including bortezomib.The treatment of melanoma cells with bortezomib was found to induce apoptosis together with the upregulation of Noxa, Mcl-1, and HSP70 proteins, and the cleavage of LC3 and autophagic formation. Also, bortezomib induced ER-stress as evidenced by the increase of intracellular Ca2+ release. In addition, bortezomib enhanced the phosphorylation of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), c-jun-N-terminal kinase (JNK) and p38, and the activation of the transcription factors AP-1, ATF-2, Ets-1, and HSF1. Bortezomib-induced mitochondrial dysregulation was associated with the accumulation of reactive oxygen species (ROS), the release of both apoptosis inducing factor (AIF) and cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of Poly (ADP-ribose) polymerase (PARP). The pretreatment of melanoma cells with the inhibitor of caspase-3 (Ac-DEVD-CHO) was found to block bortezomib-induced apoptosis that subsequently led to the increase of autophagic formation. In contrast, the inhibition of ASK1 abrogated bortezomib-induced autophagic formation and increased apoptosis induction. Furthermore, the inhibition of JNK, of HSP70 also increased apoptosis induction without influence of bortezomib-induced autophagic formation. Based on the inhibitory experiments, the treatment with bortezomib triggers the activation of both ER-stress-associated pathways, namely IRE1α–ASK1–p38–ATF-2/ets-1–Mcl-1, and IRE1α–ASK1–JNK–AP-1/HSF1–HSP70 as well as mitochondrial dysregulation-associated pathways, namely ROS–ASK1–JNK–AP-1/HSF1–HS70, and AIF–caspase-3–PARP and Cyt.c, and caspase-9–caspase-3–PARP. Taken together, our data demonstrates for the first time the molecular mechanisms, whereby bortezomib triggers both apoptosis and autophagic formation in melanoma cells.