Kathleen L. O'Connor

mTORC1 and mTORC2 regulate EMT, motility and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways

Activation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mTOR kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners toward regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor, and Rictor mRNA was noted with advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor, and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastases compared with normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition (MET) and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact and decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating epithelial-mesenchymal transition (EMT), motility, and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.

Integrin α6β4 Controls the Expression of Genes Associated with Cell Motility, Invasion, and Metastasis, Including S100A4/Metastasin

The integrin α6β4 is associated with carcinoma progression by contributing to apoptosis resistance, invasion, and metastasis, due in part to the activation of select transcription factors. To identify genes regulated by the α6β4 integrin, we compared gene expression profiles of MDA-MB-435 cells that stably express integrin α6β4 (MDA/β4) and vector-only-transfected cells (MDA/mock) using Affymetrix GeneChip® analysis. Our results show that integrin α6β4 altered the expression of 538 genes (p < 0.01). Of these genes, 36 are associated with pathways implicated in cell motility and metastasis, including S100A4/metastasin. S100A4 expression correlated well with integrin α6β4 expression in established cell lines. Suppression of S100A4 by small interference RNA resulted in a reduced capacity of α6β4-expressing cells to invade a reconstituted basement membrane in response to lysophosphatidic acid. Using small interference RNA, promoter analysis, and chromatin immunoprecipitation, we demonstrate that S100A4 is regulated by NFAT5, thus identifying the first target of NFAT5 in cancer. In addition, several genes that are known to be regulated by DNA methylation were up-regulated dramatically by integrin α6β4 expression, including S100A4, FST, PDLIM4, CAPG, and Nkx2.2. Notably, inhibition of DNA methyltransferases stimulated expression of these genes in cells lacking the α6β4 integrin, whereas demethylase inhibitors suppressed expression in α6β4 integrin-expressing cells. Alterations in DNA methylation were confirmed by bisulfate sequencing, thus suggesting that integrin α6β4 signaling can lead to the demethylation of select promoters. In summary, our data suggest that integrin α6β4 confers a motile and invasive phenotype to breast carcinoma cells by regulating proinvasive and prometastatic gene expression.

Integrin alpha6beta4 promotes expression of autotaxin/ENPP2 autocrine motility factor in breast carcinoma cells.

In advanced breast carcinomas, the α6β4 integrin is associated with a migratory and invasive phenotype. In our current study, we show that expression of the α6β4 integrin in MDA-MB-435 breast carcinoma cells leads to increased expression of the autocrine motility factor autotaxin, as determined by Affymetrix gene chip, real-time quantitative RT–PCR and immunoblot analyses. We further demonstrate that increased autotaxin secretion from integrin α6β4 expressing cells acts to enhance chemotaxis through its ability to convert lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) and accounts for 80% of the motogenic activity of the conditioned medium. We determine that integrin α6β4-dependent overexpression of autotaxin in MDA-MB-435 cells is mediated by NFAT1, but not NFAT5, through the use of siRNAs that specifically target autotaxin, integrin β4, NFAT1 and NFAT5. Finally, we show by electrophoretic mobility shift assays that two consensus NFAT binding sites found in the autotaxin promoter strongly and specifically bind NFAT1 from integrin α6β4 expressing cells. In summary, we find that the α6β4 integrin potentiates autotaxin expression through the upregulation and activation of NFAT1. These observations highlight for the first time a mechanism by which NFAT transcription factors can facilitate an invasive and motile phenotype downstream of integrin α6β4 signaling.

Inhibition of Fatty Acid Synthase Attenuates CD44-Associated Signaling and Reduces Metastasis in Colorectal Cancer

Fatty acid synthase (FASN) and ATP-citrate lyase, key enzymes of de novo lipogenesis, are significantly upregulated and activated in many cancers and portend poor prognosis. Even though the role of lipogenesis in providing proliferative and survival advantages to cancer cells has been described, the impact of aberrant activation of lipogenic enzymes on cancer progression remains unknown. In this study, we found that elevated expression of FASN is associated with advanced stages of colorectal cancer (CRC) and liver metastasis, suggesting that it may play a role in progression of CRC to metastatic disease. Targeted inhibition of lipogenic enzymes abolished expression of CD44, a transmembrane protein associated with metastases in several cancers including CRC. In addition, inhibition of lipogenic enzymes and reduced expression of CD44 attenuated the activation of MET, Akt, FAK, and paxillin, which are known to regulate adhesion, migration, and invasion. These changes were consistent with an observed decrease in migration and adhesion of CRC cells in functional assays and with reorganization of actin cytoskeleton upon FASN inhibition. Despite the modest effect of FASN inhibition on tumor growth in xenografts, attenuation of lipogenesis completely abolished establishment of hepatic metastasis and formation of secondary metastasis. Together, our findings suggest that targeting de novo lipogenesis may be a potential treatment strategy for advanced CRC.

Integrin laminin receptors and breast carcinoma progression

This review explores the mechanistic basis of breast carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression not only for their ability to mediate physical interactions with extracellular matrices but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on the α6 integrins (α6β1 and α6β4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in breast cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease. Recent work by our group and others on mechanisms of breast carcinoma invasion and survival that are influenced by the α6 integrins are discussed.

Clinical significance of the integrin α6β4 in human malignancies

Integrin α6β4 is a cellular adhesion molecule that binds to laminins in the extracellular matrix and nucleates the formation of hemidesmosomes. During carcinoma progression, integrin α6β4 is released from hemidesmosomes, where it can then signal to facilitate multiple aspects of tumor progression including sustaining proliferative signaling, tumor invasion and metastasis, evasion of apoptosis, and stimulation of angiogenesis. The integrin achieves these ends by cooperating with growth factor receptors including EGFR, ErbB-2, and c-Met to amplify downstream pathways such as PI3K, AKT, MAPK, and the Rho family small GTPases. Furthermore, it dramatically alters the transcriptome toward a more invasive phenotype by controlling promoter DNA demethylation of invasion and metastasis-associated proteins, such as S100A4 and autotaxin, and upregulates and activates key tumor-promoting transcription factors such as the NFATs and NF-κB. Expression of integrin α6β4 has been studied in many human malignancies where its overexpression is associated with aggressive behavior and a poor prognosis. This review provides an assessment of integrin α6β4 expression patterns and their prognostic significance in human malignancies, and describes key signaling functions of integrin α6β4 that contribute to tumor progression.

Dynamic functions of RhoA in tumor cell migration and invasion

RhoA is one of the more extensively studied members of the Rho family of small GTPase where it is most readily recognized for its contributions to actin-myosin contractility and stress fiber formation. Accordingly, RhoA function during cell migration has been relegated to the rear of the cell where it mediates retraction of the trailing edge. However, RhoA can also mediate membrane ruffling, lamellae formation and membrane blebbing, thus suggesting an active role in membrane protrusions at the leading edge. With the advent of fluorescence resonance energy transfer (FRET)-based Rho activity reporters, RhoA has been shown to be active at the leading edge of migrating cells where it precedes Rac and Cdc42 activation. These observations demonstrate a remarkable versatility to RhoA signaling, but how RhoA function can switch between contraction and protrusion has remained an enigma. This review highlights recent advances regarding how the cooperation of Rho effector Rhotekin and S100A4 suppresses stress fiber generation to permit RhoA-mediated lamellae formation.

The Role of Protein Kinase D in Neurotensin Secretion Mediated by Protein Kinase C-α/-δ and Rho/Rho Kinase

Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal (GI) secretion, motility, and growth as well as the proliferation of NT receptor positive cancers. Secretion of NT is regulated by phorbol ester-sensitive protein kinase C (PKC) isoforms-α and -δ and may involve protein kinase D (PKD). The purpose of our present study was: (i) to define the role of PKD in NT release from BON endocrine cells and (ii) to delineate the upstream signaling mechanisms mediating this effect. Here, we demonstrate that small interfering RNA (siRNA) targeted against PKD dramatically inhibited both basal and PMA-stimulated NT secretion; NT release is significantly increased by overexpression of PKD. PKC-α and -δ siRNA attenuated PKD activity, whereas overexpression of PKC-α and -δ enhanced PKD activity. Rho kinase (ROK) siRNA significantly inhibited NT secretion, whereas overexpression of ROKα effectively increased NT release. Rho protein inhibitor C3 dramatically inhibited both NT secretion and PKD activity. In conclusion, our results demonstrate that PKD activation plays a central role in NT peptide secretion; upstream regulators of PKD include PKC-α and -δ and Rho/ROK. Importantly, our results identify novel signaling pathways, which culminate in gut peptide release.

Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.

Upregulation and redistribution of integrin α6β4 expression occurs at an early stage in pancreatic adenocarcinoma progression

Pancreatic adenocarcinomas are highly invasive cancers for reasons that are currently unclear. Here we sought to determine if the proinvasive integrin α6β4 may be related to pancreatic adenocarcinoma tumor progression. Expression of integrin α6β4 was analyzed via immunohistochemistry for the β4 subunit in normal pancreas, pancreatic intraepithelial neoplasia (PanIN) lesions, pancreatic adenocarcinomas and chronic pancreatitis. In normal pancreatic ducts, integrin α6β4 was noted only at the cells basal interface with the basement membrane. In pancreatic adenocarcinomas, 92% (104/113) demonstrated overexpression of integrin α6β4 and altered localization to the cytoplasm and membranous regions. This pattern of expression was observed in all PanIN lesions as early as PanIN-1A, and was evident in lesions that were juxtapositioned to normal epithelium. In contrast, 93% (13/14) of chronic pancreatitis samples resembled the staining pattern of normal pancreas. When cancer was present in areas of chronic pancreatitis, this altered expression of α6β4 integrin identified the cancer. We conclude that integrin α6β4 is expressed only on the basal surface of ductal cells in normal pancreas and chronic pancreatitis. During pancreatic adenocarcinoma progression, the α6β4 integrin is dramatically overexpressed and displays altered localization at the earliest stages of PanIN, thus representing an early event in pancreatic adenocarcinoma progression.