Windy Dean-Colomb

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism

It is well known that ErbB2, a receptor tyrosine kinase, localizes on the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through the association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to ErbB2 targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Panepoxydone Targets NF-kB and FOXM1 to Inhibit Proliferation, Induce Apoptosis and Reverse Epithelial to Mesenchymal Transition in Breast Cancer

Background Triple-negative breast cancer (TNBC) is a highly diverse group that is associated with an aggressive phenotype. Its treatment has been challenging due to its heterogeneity and absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. NF-κB is over-expressed in many breast cancers; thus, inactivation of the NF-κB pathway could serve as a therapeutic target. Here we report for the first time the anti-tumor activity of panepoxydone (PP), a NF-κB inhibitor isolated from an edible mushroom, in several breast cancer cell lines. Methods We investigated the effects of PP on cell growth, migration-invasion, apoptosis and EMT-related proteins expression in MCF-7 and TNBC cell lines MDA-MB-231, MDA-MB-468 and MDA-MB-453. Results Significant antitumor activity was seen in all cell lines, with differential responses noted in cell-line specific manner. Treatment with PP resulted in significant cytotoxicity, decreased invasion, migration and increased apoptosis in all cell lines tested. Up-regulation of Bax and cleaved PARP and down-regulation of Bcl-2, survivin, cyclin D1 and caspase 3 were noted in PP-treated breast cancer cells. The antitumor effect of PP appeared related to its ability to inhibit the phosphorylation of inhibitor of NF-κB (IκBα) with cytoplasmic accumulation. PP treatment also down-regulated FOXM1 which resulted in a reversal of EMT. Similar results were obtained after silencing of NF-kB and FOXM1. Conclusion Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. These results suggest that the PP may be a potentially effective chemopreventive or therapeutic agent against breast cancer. However, additional studies are required to more fully elucidate the mechanism of antitumor effect of PP.

Kaiso, a transcriptional repressor, promotes cell migration and invasion of prostate cancer cells through regulation of miR-31 expression

Kaiso, a member of the BTB/POZ zinc finger protein family, functions as a transcriptional repressor by binding to sequence-specific Kaiso binding sites or to methyl-CpG dinucleotides. Previously, we demonstrated that Kaiso overexpression and nuclear localization correlated with the progression of prostate cancer (PCa). Therefore, our objective was to explore the molecular mechanisms underlying Kaiso-mediated PCa progression. Comparative analysis of miRNA arrays revealed that 13 miRNAs were significantly altered (> 1.5 fold, p < 0.05) in sh-Kaiso PC-3 compared to sh-Scr control cells. Real-time PCR validated that three miRNAs (9, 31, 636) were increased in sh-Kaiso cells similar to cells treated with 5-aza-2′-deoxycytidine. miR-31 expression negatively correlated with Kaiso expression and with methylation of the miR-31 promoter in a panel of PCa cell lines. ChIP assays revealed that Kaiso binds directly to the miR-31 promoter in a methylation-dependent manner. Over-expression of miR-31 decreased cell proliferation, migration and invasiveness of PC-3 cells, whereas cells transfected with anti-miR-31 restored proliferation, migration and invasiveness of sh-Kaiso PC-3 cells. In PCa patients, Kaiso high/miR-31 low expression correlated with worse overall survival relative to each marker individually. In conclusion, these results demonstrate that Kaiso promotes cell migration and invasiveness through regulation of miR-31 expression.

Abstract 5571: Antitumor activity of a novel natural therapeutic agent against triple negative breast cancer.

Purpose: Triple-negative breast cancer (TNBC), representing up to 20% of all breast cancer cases, is a highly diverse group of cancer that is associated with an aggressive phenotype, with affected patients having a poorer prognosis. Its treatment has been challenging due to its heterogeneity and the absence of well-defined molecular targets. Thus, there is an urgent need to identify novel agents with therapeutic application. Nuclear factor kappa-B (NF-κB), a transcription factor, has been shown to be significantly increased in TNBC, consistent with the aggressiveness of these tumors. Thus, inactivation of the NF-κB pathway, could serve as therapeutic targets for treatment of TNBC. Panepoxydone (PP), a compound isolated from Lentinus crinitus (an edible mushroom), has been shown to interfere with NF-κB mediated signal transduction by inhibiting the phosphorylation of IκBα. Here we evaluate the antitumor activity of panepoxydone in several breast cancer cell lines. Experimental Design: Estrogen receptor positive (MCF-7) and three triple negative breast cancer cell line subtypes (MDAMB-231, MDAMB-468 and MDAMB-453) were treated with increasing concentrations of panepoxydone or DMSO (final concentration, 0.2%). Results: Significant antitumor activity was seen in all the breast cancer cell lines, with differential responses noted in a cell-line specific manner. Whereas treatment with PP resulted in significant cytotoxicity, along with decreased invasion and migration in all the cell lines tested, with IC50 of 4-15 μM, MDAMB-453 cells were the most sensitive, whereas MDAMB-231 were almost 4-fold less sensitive. PP treatment also induced apoptosis in a cell-line specific manner, with minimal effect noted in MCF7 and MDAMB-231 cells, but a 35.8% and 40.8% increase in apoptotic cells noted in MDAMB-453 and MDAMB-468 cells, respectively. Differential response to PP treatment was also noted in the decreased expression of the apoptosis-related proteins Bcl-2, survivin, and cyclin D1, with MDAMB-453 again having at least a 3-fold greater decrease in expression when compared to the other TNBC cell lines. The antitumor effect of PP, appeared in part, to be related to its ability to inhibit phosphorylated IκBα, with accumulation of IκBα noted in a dose-dependent manner in all the cell lines. This resulted in reduced translocation of NF-kB from cytoplasm to nucleus as analyzed via immunofluorescence. Conclusion: Altogether, these studies show, for the first time the antitumor activity of PP against breast cancer cells, in particular TNBC cells. Furthermore, it highlights the concept that optimal treatment of TNBC warrants attention to the differential sensitivity of various TNBC subtypes to therapeutic agents. Additional studies are required to further elucidate the antitumor effect of panepoxydone, but these studies provide support for the further evaluation of PP as a therapeutic agent in the treatment of TNBC. Citation Format: Ritu Arora, Bernard D. Gary, Steven McClellan, Yaguang Xi, Gary Piazza, Eddie Reed, Laurie Owen, Windy Dean-Colomb. Antitumor activity of a novel natural therapeutic agent against triple negative breast 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 5571. doi:10.1158/1538-7445.AM2013-5571

Abstract 3370: Natural compound targeting metabolism: A new insight for the treatment of triple negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Triple-negative breast cancer (TNBC) is a highly diverse group associated with an aggressive phenotype, characterized by early relapse and chemotherapy as the only treatment option. Thus, there is an urgent need to identify alternative strategies for the treatment of TNBC. Use of aerobic glycolysis is important marker of cancer cells and thus monitoring and targeting metabolism in cancer cells is an expanding area of research. Lactate dehydrogenase (LDH) A and B are important enzymes in the metabolic pathway. Traditionally, LDH-A expression has been used to measure the glycolytic capacity of cancer cells however, more recently LDH-B has also been identified as an important biomarker of metabolism. LDH-A is over-expressed in breast cancer, while presence of LDH-B in tumors is contradictory. Higher expression of LDH-B has been reported in normal tissues, with lower expression also noted in breast and prostate tumors. Thus, clarification of the role of LDH-A and LDH-B in cancer metabolism is important in identifying potential targets for TNBC treatment. NF-κB plays an important role in modulating cancer metabolism through regulating the balance between glycolysis and mitochondrial respiration and is over-expressed in TNBCs. Thus, the focus of the present study is to determine the effect of an NF-KB inhibitor in altering cellular metabolism of aggressive breast cancer cells. Experimental Design: We measured the metabolic capacity of several breast cancer cell lines via measurement of mitochondrial membrane potential, ATP level and expression of LDH-A and LDH-B. We then evaluated the ability of the NF-kB inhibitor, panepoxydone (PP), to alter these markers of metabolism. Results: MCF-7 and MDA-MB-231 cells showed dose-dependent decreases in mitochondrial membrane potential, in the form of increased mitochondrial damage, along with decreased ATP levels, when treated with the NF-kB inhibitor, PP. Immunoblotting of LDH-A and LDH-B expression in MCF-7 and MDA-MB-231 cells showed high expression of LDH-A in both cell lines, while MDA-MB-231 cells had a higher level of LDH-A compared to LDH-B, MCF-7 cells had no LDH-B. When these cells were treated with PP, LDH-A levels was reduced in both cancer cell lines (3.5 -4 fold). Interestingly, elevated level of LDH-B was noticed in MDA-MB-231 cells (2 fold). We used Human mammary epithelial cells (HMEC) to check the basal level of LDH-A and LDH-B and almost similar level was observed. Furthermore, this increase in LDH-B was associated with decreased aggressiveness, as noted by decreased invasion and migration. Conclusion: All together this data highlights the role of targeting NF-kB in altering glycolysis through modulation of the membrane potential, ATP level and LDH-A and LDH-B levels. Additionally, it appears that simultaneous measurement, of both LDH-A and LDH-B, may be a more accurate predictor of the metabolic capacity of cancer cells and thus a reflection of their aggressiveness. Citation Format: Ritu Arora, David Schmitt, Steve McClellan, Ming Tan, Windy Dean-Colomb. Natural compound targeting metabolism: A new insight for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2014-3370

Abstract P1-07-07: Targeting reversal of epithelial-mesenchymal transition through FOXM1 inhibition in breast cancer cells

Purpose: Triple-negative breast cancer (TNBC), representing up to 20% of all breast cancer cases, is a highly diverse group of cancer that is associated with an aggressive phenotype, with affected patients having a poorer prognosis. Its treatment has been challenging due to its heterogeneity and the absence of well-defined molecular targets. Epithelial-to-mesenchymal transition (EMT) is one of the hallmarks of aggressive breast cancers and is associated with increased metastatic potential. TNBCs have been shown to exhibit elevated EMT. Thus, reversal of EMT has been identified as a viable target in the treatment of aggressive breast cancers. FOXM1 is an oncogenic transcription factor of the Forkhead family and it has a well-defined role in cell proliferation and cell-cycle progression. FOXM1 has been reported to be over-expressed in breast cancer. In prostate cancer, FOXM1 overexpression has been linked to increase EMT. We have previously shown that targeting NF-kB with panepoxydone (PP), an NF-kB inhibitor isolated from Lentinus crinitus (an edible mushroom), has significant antitumor activity against breast cancer cells with a resultant decrease in cell migration and invasion and induction of apoptosis. We have further identified that the FOXM1 promoter has a binding site for NF-kB. Thus, in this study, we investigated whether PP-induced inhibition of breast cancer cell growth could be attributed to FoxM1 activity and EMT reversal. Experimental Design: The estrogen receptor positive (MCF-7) and three different triple negative (MDA-MB-231, MDA-MB-453, and MDA-MB-468) breast cancer cell lines were selected to observe the effect of PP on FOXM1and EMT markers. Results: In the current study, we investigated whether panepoxydone- induced inhibition of breast cancer cell growth could be attributed to FoxM1activity and EMT reversal. Breast cancer cells were treated with increasing concentrations of PP and proteins collected after 24 hrs. Panepoxydone treatment significantly reduced FOXM1 expression in MCF-7 and MDA-MB-231 (1.7 and 2.4-fold, respectively, p To further elucidate the relationship between FOXM1 on EMT reversal, we then silenced FOXM1 in MDA-MB-231 cells and evaluated its effect on the EMT-associated markers. Again we noted upregulation of E cadherin (3.5-fold, p Conclusion: These studies show, for the first time, the role of FOXM1 in EMT reversal in breast cancer cells. Furthermore, it shows that use of an NF-kB inhibitor, such as panepoxydone, may be helpful in elucidating the relationship between these two transcriptional factors, in the reversal of the EMT phenotype in aggressive breast cancer cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-07.

Abstract 3025: Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism.

It is well accepted that ErbB2, an important oncoprotein, localizes on the plasma membrane as a receptor tyrosine kinase. This study describes a novel observation that ErbB2 localizes in mitochondria of multiple cancer cell lines and patient samples. We identified an endogenous mitochondrial targeting sequence in ErbB2 and found that ErbB2 translocates into mitochondria through the association with mtHSP70, a key player in the canonical mitochondrial protein importation pathway. Additionally, we observed that mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complex I, II and IV of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased by mtErbB2. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Mitochondrial ErbB2 regulates the phosphorylation and activity of COX II and consequently the cytochrome c release and apoptosis. Additional studies showed that cancer cells with higher levels of mtErbB2 were more resistant to Trastuzumab and deletion of the mitochondrial targeting sequence of ErbB2 sensitized the cells to Trastuzumab. Our study provides a novel perspective on the metabolic regulatory functions of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. Since ErbB2 plays important roles in multiple organs and multiple types of cancers, the study may have broad impact on the fields of cancer biology. The work was supported by The Vincent F. Kilborn Jr. Cancer Research Foundation and National Institutes of Health Grant RO1CA149646. Citation Format: Yan Ding, Zixing Liu, Shruti Desai, Lewis Pannell, Hong Yi, Elizabath Wright, Laurie Owen, Windy Dean-Colomb, Oystein Fodstad, Jianrong Lu, Ming Tan. Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism. [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 3025. doi:10.1158/1538-7445.AM2013-3025