Andrei V. Bakin

Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration.

We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt signaling in transforming growth factor β (TGFβ)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGFβ1 induced phosphorylation of Akt at Ser-473 and Akt in vitro kinase activity against GSK-3β within 30 min. These responses were temporally correlated with delocalization of E-cadherin, ZO-1, and integrin β1 from cell junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-negative mutant of Akt blocked the delocalization of ZO-1 induced by TGFβ1, whereas transfection of constitutively active p110 induced loss of ZO-1 from tight junctions. In addition, LY294002 blocked TGFβ-mediated C-terminal phosphorylation of Smad2. Consistent with these data, TGFβ-induced p3TP-Lux and p(CAGA)12-Lux reporter activities were inhibited by LY294002 and transiently expressed dominant-negative p85 and Akt mutants in NMuMG and 4T1 cells. Dominant-negative RhoA inhibited TGFβ-induced phosphorylation of Akt at Ser-473, whereas constitutively active RhoA increased the basal phosphorylation of Akt, suggesting that RhoA in involved in TGFβ-induced EMT. Finally, LY294002 and neutralizing TGFβ1 antibodies inhibited ligand-independent constitutively active Akt as well as basal and TGFβ-stimulated migration in 4T1 and EMT6 breast tumor cells. Taken together, these data suggest that PI3K-Akt signaling is required for TGFβ-induced transcriptional responses, EMT, and cell migration.

PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization.

We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27kip1. Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor p27Kip1. Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT157D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser473 phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.

Autocrine Transforming Growth Factor-β Signaling Mediates Smad-independent Motility in Human Cancer Cells

Transforming growth factor-β (TGF-β) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-β-mediated growth inhibition, autocrine TGF-β signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-β may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-β receptor (TβRII-K277R). Our data indicate that TβRII-K277R is expressed, can associate with the type I TGF-β receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-β. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of TβRII-K277R expressing cells was impaired. The impaired motility of TβRII-K277R cells could be restored by reconstituting TGF-β signaling with a constitutively active TGF-β type I receptor (ALK5TD) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5TD expression sufficient to restore motility in the cells expressing TβRII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-β activation and suggest that TGF-β promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.

Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion

Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (ρ0 cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and ρ0 epithelial cells. The focal proteins in these networks include i) FN1 (fibronectin) and ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 5 of 6 members of peroxiredoxin whose expression were altered in ρ0 epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect the chromosomal stability. Consistent with above finding our study revealed an increase DNA double strand breaks, and unique chromosomal rearrangements in ρ0 breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that i) claudin-1 and 7 were indeed down regulated in ρ0 breast epithelial cells, ii) down regulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and iii) claudin-1 and -7 were also down regulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells

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.

ALK5 promotes tumor angiogenesis by upregulating matrix metalloproteinase-9 in tumor cells.

Transforming growth factor beta 1 (TGF-β1) is a potent tumor suppressor but, paradoxically, TGF-β1 enhances tumor growth and metastasis in the late stages of cancer progression. This study investigated the role of TGF-β type I receptor, ALK5, and three mitogen-activated protein kinases (MAPKs) in metastasis by breast cancer cell line MDA-MB-231. We show that autocrine TGF-β signaling in MDA-MB-231 cells is required for tumor cell invasion and tumor angiogenesis. Expression of kinase-inactive ALK5 reduces tumor invasion and formation of new blood vessels within the tumor orthotopic xenografts in severe combined immunodeficiency (SCID) mice. In contrast, constitutively active ALK5-T204D enhances tumor invasion and angiogenesis by stimulating expression of matrix metalloproteinase MMP-9/gelatinase-B. Ablation of MMP-9 in ALK5-T204D cells by RNA interference (RNAi) reduces tumor invasion and tumor growth. Importantly, RNAi-MMP-9 reduces tumor neovasculature and increases tumor cell death. Induction of MMP-9 by TGF-β-ALK5 signaling requires MEK-ERK but not JNK, p38 MAPK or Smad4. Dominant-negative MEK blocks and constitutively active MEK1 enhances MMP-9 expression. However, all three MAPK cascades (ERK, JNK and p38 MAPK) are required for TGF-β-mediated cell migration. Collectively, our results show that TGF-β-ALK5-MAPK signaling in tumor cells promotes tumor angiogenesis and MMP-9 is an important component of this program.

Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-beta.

Loss of actin stress fibers has been associated with cell transformation and metastasis. TGF-β induction of stress fibers in epithelial cells requires high molecular weight tropomyosins encoded by TPM1 and TPM2 genes. Here, we investigated the mechanism underlying the failure of TGF-β to induce stress fibers and inhibit cell migration in metastatic cells. RT–PCR analysis in carcinoma cell lines revealed a significant reduction in TPM1 transcripts in metastatic MDA-MB-231, MDA-MB-435 and SW620 cell lines. Treatment of these cells with demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) increased mRNA levels of TPM1 with no effect on TPM2. Importantly, 5-aza-dC treatment of MDA-MB-231 cells restored TGF-β induction of TPM1 and formation of stress fibers. Forced expression of TPM1 by using Tet-Off system increased stress fibers in MDA-MB-231 cells and reduced cell migration. A potential CpG island spanning the TPM1 proximal promoter, exon 1, and the beginning of intron 1 was identified. Bisulfite sequencing showed significant cytosine methylation in metastatic cell lines that correlated with a reduced expression of TPM1. Together these results suggest that epigenetic suppression of TPM1 may alter TGF-β tumor suppressor function and contribute to metastatic properties of tumor cells.

TAK1 is required for TGF-β1-mediated regulation of matrix metalloproteinase-9 and metastasis

Transforming growth factor-β 1 (TGF-β1) signaling in tumor cells has been implicated in tumor angiogenesis and metastasis by regulating matrix proteolysis. Although MMP-9/gelatinase-B is an important component of these TGF-β1 responses, the mechanism of its regulation is not well understood. Here, we present evidence that TGF-β-activated protein kinase 1 (TAK1) is critical for TGF-β regulation of MMP-9 and the metastatic potential of breast cancer cell line MDA-MB-231. We found that suppression of TAK1 signaling by dominant-negative (dn) TAK1 or RNA interference (siRNA) reduces expression of MMP-9 and tumor cell invasion, without growth inhibition in cell culture. The orthotopic xenograft studies in SCID mice showed that suppression of TAK1 signaling by dn-TAK1 reduces tumor growth and formation of lung metastases. Dn-TAK1 reduced the proliferation Ki-67 index and neovasculature of orthotopic xenografts. TAK1-mediated regulation of MMP-9 involves NF-κB signaling. Dn-TAK1 reduces NF-κB transcriptional response and inhibition of NF-κB reduces expression of MMP-9 and activity of the MMP-9 promoter reporter. Together, these findings suggest that TAK1 contributes to TGF-β1-mediated tumor angiogenesis and metastasis via a mechanism involving the TAK1–NF-κB–MMP-9 pathway.

Integrin β5 contributes to the tumorigenic potential of breast cancer cells through the Src-FAK and MEK-ERK signaling pathways

Cancer progression, response to therapy and metastasis depend on tumor microenvironment. Integrins are cell-adhesion receptors that mediate interactions of cells with extracellular matrix. The αv-β-family of integrins contributes to tumorigenesis, response to therapy and cancer stem cell biology. Thus, understanding the function of specific integrins in cancer is critical for the development of therapeutic approaches targeting integrins. The study investigated the role of integrin β5 in breast carcinomas by depleting integrin β5 using RNA interference and reexpression of integrin β5. Depletion of integrin β5 in triple-negative breast carcinoma cells markedly reduced tumor take, growth and tumor angiogenesis, whereas reexpression of integrin β5 rescued this phenotype. Reduction in tumor angiogenesis is associated with lower expression of vascular endothelial growth factor-A in integrin β5-depleted tumors. Tumor cells deficient in integrin β5 have lower migration and proliferative capacities. Biochemical assays revealed that integrin β5 mediates the Src-focal adhesion kinase and MEK-extracellular signal-regulated kinase signaling events that operate independently, and inhibition of these pathways phenocopies integrin β5 deficiency. Breast carcinoma cells express high levels of integrin β5, whereas expression of integrin β3 is limited to stromal compartments and integrin β6 is lost in metastatic cells. Together, these findings show a critical role for integrin β5 in the tumorigenic potential of breast carcinoma cells and therapeutic targeting of integrin β5 is especially attractive for triple-negative breast carcinomas, which are refractory to most of the current therapies.

Role of high‐molecular weight tropomyosins in TGF‐β‐mediated control of cell motility

Transforming growth factor beta1 (TGF‐β1) suppresses tumor development at early stages of cancer, but enhances tumor invasion and formation of metastasis. TGF‐β1‐mediated tumor invasion is associated with epithelial to mesenchymal transition (EMT) and matrix proteolysis. The mechanisms of these TGF‐β1 responses in normal and tumor cells are not well understood. Recently, we have reported that TGF‐β1 increases expression of high‐molecular weight tropomyosins (HMW‐tropomyosins) and formation of actin stress fibers in normal epithelial cells. The present study investigated the role of tropomyosin in TGF‐β1‐mediated cell motility and invasion. We found that TGF‐β1 restricts motility of normal epithelial cells although it promotes EMT and formation of actin stress fibers and focal adhesions. Cell motility was enhanced by siRNA‐mediated suppression of HMW‐tropomyosins. TGF‐β1 stimulated migration and matrix proteolysis in breast cancer MDA‐MB‐231 cells that express low levels of HMW‐tropomyosins. Tet‐Off‐regulated expression of HMW‐tropomyosin inhibited cell migration and matrix proteolysis without affecting expression of matrix metalloproteinases. Tropomyosin increased cell adhesion to matrix by enhancing actin fibers and focal adhesions. Finally, tropomyosin impaired the ability of tumor cells to form lung metastases in SCID mice. Thus, these results suggest that HMW‐tropomyosins are important for TGF‐β‐mediated control of cell motility and acquisition of the metastatic potential.