Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin

The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2

TGF-β signaling promotes tumor vasculature by enhancing the pericyte-endothelium association

Abstract Background The breast cancer microenvironment promotes tumor vascularization through the complex interactions involving tumor-associated fibroblasts (TAFs). Emerging data indicate that TAFs increase production and signaling by TGF-β cytokines, while the role of TGF-β signaling in the regulation of tumor blood vessels is not fully understood. The current study presents evidence that TAFs enhance the organization of tumor blood capillaries, and TGF-β signaling plays an important role in this response. Methods Tumor vascularization was studied in xenograft models of breast carcinoma cells, alone and in combination with fibroblasts. TGF-β signaling in breast cancer cells was modulated by expression of kinase-inactive TGFBR1-K232R (dnTGFBR1) or constitutive-active TGFBR1-T204D (caTGFBR1) receptor mutants. The architecture of tumor blood capillaries was assessed by immune-histochemical analysis of endothelium and pericytes. The role of TGF-β-Smad signaling in fibronectin expression was examined using adenoviral transduction of signaling components. Results Our studies revealed that TAFs significantly increase the lumen size of blood microvessels. Inactivation of TGF-β signaling in tumor cells by dnTGFBR1 reduced the microvessel density and lumen sizes, decreasing tumor growth. In contrast, caTGFBR1-tumors exhibited greater vessel density and lumen sizes. Tumors with inactive dnTGFBR1 showed lower amounts of TAFs, while caTGFBR1 increased amounts of TAFs compared to the control. Inspection of pericytes and endothelial cells in tumor vasculature revealed that TAFs enhanced vessel coverage by pericytes, vascular cells supporting capillaries. This effect was impaired in dnTGFBR1-tumors, whereas active caTGFBR1 enhanced the association of pericytes with endothelium. Accordingly, dnTGFBR1-tumors exhibited the presence of hemorrhages, a sign of fragile blood vessels. Biochemical analysis showed that TGFBR1-SMAD signaling up-regulates fibronectin, a prominent regulator of endothelium-pericyte interactions. Conclusions The current study indicates that tumor-fibroblast crosstalk enhances tumor vascularization by increasing the pericyte-endothelium association via a mechanism involving the TGFβ-fibronectin axis. The tumor-fibroblast model represents a useful system for dissecting the complex interactions governing tumor angiogenesis and developing new approaches to therapeutic targeting tumor vasculature

TGF-beta autocrine pathway and MAPK signaling promote cell invasiveness and in vivo mammary adenocarcinoma tumor progression

Breast cancer progression and metastasis have been linked to abnormal signaling by transforming growth factor-beta (TGF-beta) cytokines. In early-stage breast cancers, TGF-beta exhibits tumor suppressor activity by repressing cell proliferation and inducing cell death, whereas in advanced-stage tumors, TGF-beta promotes invasion and metastatic dissemination. The molecular mechanisms underlying pro-oncogenic activities of TGF-beta are not fully understood. The present study validates the role of TGF-beta signaling in cancer progression and explores mediators of pro-oncogenic TGF-beta activities using the LM3 mammary adenocarcinoma cell line, derived from a spontaneous murine mammary adenocarcinoma. Expression of kinase-inactive TGF-beta receptors decreased both basal and TGF-beta-induced invasion. Analysis of signal transduction mediators showed that p38MAPK and MEK contribute to TGF-beta stimulation of cell motility and invasion. TGF-beta disrupted the epithelial actin structures supporting cell-cell adhesions, and increased linear actin filaments. Moreover, MEK and p38MAPK pathways showed opposite effects on actin remodeling in response to TGF-beta. Blockade of Raf-MEK signaling enhanced TGF-beta induction of actin stress-fibers whereas p38MAPK inhibitors blocked this effect. A novel observation was made that TGF-beta rapidly activates the actin nucleation Arp2/3 complex. In addition, TGF-beta stimulated matrix metalloproteinase MMP-9 secretion via a MAPK-independent pathway. Experiments using syngeneic mice showed that kinase-inactive TGF-beta receptors inhibit the first stages of LM3 tumor growth in vivo. Our studies demonstrate that autocrine TGF-beta signaling contributes to the invasive behavior of mammary carcinoma cells. Moreover, we show that both MAPK-dependent and -independent pathways are necessary for TGF-beta-induced effects. Therefore, MEK-ERK and p38 MAPK pathways are potential venues for therapeutic intervention in pro-oncogenic TGF-beta signaling.Fil: Daroqui, Maria Cecilia. Montefiore Medical Center; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Paula. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; ArgentinaFil: Bal, Elisa Dora. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bakin, Andrei V.. Roswell Park Cancer Institute; Estados UnidosFil: Puricelli, Lydia I.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncologia "Angel H. Roffo"; Argentin

TGF-β autocrine pathway and MAPK signaling promote cell invasiveness and in vivo mammary adenocarcinoma tumor progression

Breast cancer progression and metastasis have been linked to abnormal signaling by transforming growth factor-β (TGF-β) cytokines. In early-stage breast cancers, TGF-β exhibits tumor suppressor activity by repressing cell proliferation and inducing cell death, whereas in advanced-stage tumors, TGF-β promotes invasion and metastatic dissemination. The molecular mechanisms underlying pro-oncogenic activities of TGF-β are not fully understood. The present study validates the role of TGF-β signaling in cancer progression and explores mediators of pro-oncogenic TGF-β activities using the LM3 mammary adenocarcinoma cell line, derived from a spontaneous murine mammary adenocarcinoma. Expression of kinase-inactive TGF-β receptors decreased both basal and TGF-β-induced invasion. Analysis of signal transduction mediators showed that p38MAPK and MEK contribute to TGF-β stimulation of cell motility and invasion. TGF-β disrupted the epithelial actin structures supporting cell-cell adhesions, and increased linear actin filaments. Moreover, MEK and p38MAPK pathways showed opposite effects on actin remodeling in response to TGF-β. Blockade of Raf-MEK signaling enhanced TGF-β induction of actin stress-fibers whereas p38MAPK inhibitors blocked this effect. A novel observation was made that TGF-β rapidly activates the actin nucleation Arp2/3 complex. In addition, TGF-β stimulated matrix metalloproteinase MMP-9 secretion via a MAPK-independent pathway. Experiments using syngeneic mice showed that kinase-inactive TGF-β receptors inhibit the first stages of LM3 tumor growth in vivo. Our studies demonstrate that autocrine TGF-β signaling contributes to the invasive behavior of mammary carcinoma cells. Moreover, we show that both MAPK-dependent and -independent pathways are necessary for TGF-β-induced effects. Therefore, MEK-ERK and p38 MAPK pathways are potential venues for therapeutic intervention in pro-oncogenic TGF-β signaling

XBP1-KLF9 Axis Acts as a Molecular Rheostat to Control the Transition from Adaptive to Cytotoxic Unfolded Protein Response

Summary: Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress. : The transcription factor XBP1s plays a central role in suppression of endoplasmic reticulum (ER) stress through direct activation of multiple genes controlling protein refolding. Fink et al. report that elevation of ER stress above a certain threshold triggers an XBP1s-dependent transcriptional program, leading to exacerbation of ER stress and cell death. Keywords: endoplasmic reticulum stress, XBP1s, KLF9, TMEM38B, ITPR1, calcium channel, UP

Down-Regulation of WAVE3, a Metastasis Promoter Gene, Inhibits Invasion and Metastasis of Breast Cancer Cells

The expression of WAVE3, an actin-cytoskeleton and reorganization protein, is elevated in malignant human breast cancer, yet the role of WAVE3 in promoting tumor progression remains undefined. We have recently shown that knockdown of WAVE3 expression in human breast adenocarcinoma MDA-MB-231 cells using small interfering RNA resulted in a significant reduction of cell motility, migration, and invasion, which correlated with a reduction in the levels of active p38 mitogen-activated protein kinase. Here, we investigated the effect of stable suppression of WAVE3 by short hairpin RNA on tumor growth and metastasis in xenograft models. Breast cancer MDA-MB-231 cells expressing short hairpin RNA to WAVE3 (shWAVE3) showed a significant reduction in Matrigel invasion and formation of lung colonies after tail-vein injection in SCID mice. In the orthotopic model, we observed a reduction in growth rate of the primary tumors, as well as in the metastases to the lungs. We also show that suppression of p38 mitogen-activated protein kinase activity by dominant-negative p38 results in comparable phenotypes to the knockdown of WAVE3. These studies provide direct evidence that the WAVE3-p38 pathway contributes to breast cancer progression and metastasis