Tumor Cell Anchorage-independent Growth Research Articles

Abstract B16: Role of RasGRF2 in AnxA6-mediated growth of TNBC cells

Abstract Introduction: The mechanisms by which annexin A6 (AnxA6) inhibits anchorage-independent tumor cell growth but promotes tumor cell motility remain unclear. Using whole-genome gene expression profiling, we identified RasGRF2 (GRF2), a Ca2+-activated RasGEF, as one of the most upregulated genes following downregulation of AnxA6 in breast cancer (BC) cells. Here, we examined the role of GRF2 as a critical RasGEF in AnxA6-meditated BC cell growth. Methods and Results: We show that the cellular levels of GRF2 are relatively low in AnxA6-high mesenchymal-like BC cells but higher in AnxA6-low basal-like BC cells. We also show that loss of AnxA6 in AnxA6-high BC cells is associated with early onset and rapid growth of xenograft tumors, while upregulation of AnxA6 in AnxA6-low BC cells is associated with a decrease in growth of xenograft tumors. To explore the reciprocal expression of GRF2 and AnxA6, we demonstrate that ionomycin-induced surge in intracellular Ca2+ led to GRF2 downregulation and that AnxA6, GRF2 and Ras proteins are within the same complex. We demonstrate that upon activation of EGFR, GRF2 is rapidly degraded and that the basal GRF2 levels were restored as the receptor activity decreased. The activation/degradation of GRF2 was followed by the activation of ERK1/2, was not affected by chelation of intracellular Ca2+ with BAPTA, but GRF2 was stabilized by chelation of extracellular Ca2+ with EGTA. Lapatinib treatment efficiently blocked the activation of EGFR but did not block the degradation of GRF2. Finally, we show that downregulation of GRF2 and inhibition of EGFR led to a greater inhibition of tumor cell growth. Conclusion: Together, these data identify RasGRF2 as a major effector of AnxA6-dependent breast tumor growth and that reduced cellular levels of GRF2 imply active RasGEF. Combined inhibition of GRF2 and EGFR may be a viable alternative treatment for triple-negative BC. Citation Format: Diva S. Whalen, Sarrah E. Widatalla, Olga Y. Korolkova, Stephen D. Williams, Heather K. Beasley, Amos M. Sakwe. Role of RasGRF2 in AnxA6-mediated growth of TNBC cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B16.

Mek activity is required for ErbB2 expression in breast cancer cells detached from the extracellular matrix.

Detachment of non-malignant epithelial cells from the extracellullar matrix (ECM) triggers their growth arrest and apoptosis. Conversely, carcinoma cells can grow without adhesion to the ECM. This capacity for anchorage-independent growth is thought to be critical for tumor progression. ErbB2/Her2 oncoprotein is overproduced by a significant fraction of breast cancers and promotes anchorage-independent tumor cell growth by poorly understood mechanisms. In an effort to understand them we found that in order to produce ErbB2, detached breast cancer cells require the activity of an ErbB2 effector protein kinase Mek and that Mek-driven ErbB2 expression is neccesary for anchorage-independent growth of such cells. We observed that Mek inhibition does not alter ErbB2 mRNA levels in detached cancer cells and that ErbB2 protein loss induced by this inhibition can be blocked by a lysosomal inhibitor. We also noticed that an increase of the density of cancer cells detached from the ECM downregulates a Mek effector protein kinase Erk and causes ErbB2 loss. Those cells that survive after ErbB2 loss display resistance to trastuzumab, an anti-ErbB2 antibody used for ErbB2-positive breast cancer treatment. Thus, Mek-induced ErbB2 stabilization in detached breast cancer cells is critical for their ability to grow anchorage-independently and their trastuzumab sensitivity.

AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin.

Loss of epithelial polarity impacts organ development and function; it is also oncogenic. AMPK, a key sensor of metabolic stress stabilizes cell-cell junctions and maintains epithelial polarity; its activation by Metformin protects the epithelial barrier against stress and suppresses tumorigenesis. How AMPK protects the epithelium remains unknown. Here, we identify GIV/Girdin as a novel effector of AMPK, whose phosphorylation at a single site is both necessary and sufficient for strengthening mammalian epithelial tight junctions and preserving cell polarity and barrier function in the face of energetic stress. Expression of an oncogenic mutant of GIV (cataloged in TCGA) that cannot be phosphorylated by AMPK increased anchorage-independent growth of tumor cells and helped these cells to evade the tumor-suppressive action of Metformin. This work defines a fundamental homeostatic mechanism by which the AMPK-GIV axis reinforces cell junctions against stress-induced collapse and also provides mechanistic insight into the tumor-suppressive action of Metformin.

Abstract 1221: Pontin, a new mutant p53 binding protein, promotes gain-of-function of mutant p53

Abstract Tumor suppressor p53 is frequently mutated in human cancers. Many tumor-associated mutant p53 (mutp53) proteins gain new functions in promoting tumorigenesis, defined as gain of function (GOF). The mechanisms for mutp53 GOF are not well-understood. Mutp53 has been reported to interact with some proteins and it has been suggested that some interactions of mutp53 with its binding partners are important for mutp53 GOF through regulating the levels or functions of mutp53 and/or its binding proteins. To understand the molecular mechanisms for mutp53 GOF, we searched for mutp53 binding proteins in thymic lymphomas of R172H mutp53 knock-in (p53R172H/R172H) mice with significant mutp53 protein accumulation by employing an unbiased immunoprecipitation (IP) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach. Normal tissues of p53R172H/R172H mice with low mutp53 levels and tumors of p53-/- mice were used as controls. Using this approach, we identified Pontin, a highly conserved AAA+ ATPase important for various cellular functions, as a new mutp53 binding protein. This Pontin-mutp53 is conserved in both humans and mice. Importantly, this Pontin-mutp53 interaction promotes mutp53 GOF in invasion, migration, and anchorage-independent growth of tumor cells. Furthermore, we found that the C-terminus of Pontin is required for Pontin-mutp53 interaction and mutp53 GOF. The ATPase domain in the C-terminus of Pontin is crucial for its promoting effect on mutp53 GOF; blocking the ATPase activity of Pontin by a Pontin specific ATPase inhibitor or an ATPase deficient dominant-negative Pontin expression vector greatly diminished mutp53 GOF. We found that Pontin was largely co-localized with mutp53 in the nucleus and promotes mutp53 GOF through regulation of mutp53 transcriptional activity; knock-down of Pontin abolished the transcriptional regulation of mutp53 towards a group of genes involved in many important signaling pathways, including gap junction signaling, IGF-1 signaling, EGF signaling etc. Furthermore, overexpression of Pontin is associated with poor survival in cancer patients, especially those containing mutp53 in tumors. This work demonstrates that Pontin promotes mutp53 GOF in tumorigenesis in an ATPase-dependent manner, and raises interesting and intriguing possibilities to block mutp53 GOF in tumors through targeting Pontin or its ATPase activity. In summary, our results highlight an important role and mechanism for Pontin, a new p53 partner, in promoting mutp53 GOF in tumorigenesis. Citation Format: Yuhan Zhao, Cen Zhang, Xuetian Yue, Xiaoyan Li, Juan Liu, Haiyang Yu, Qifeng Yang, Zhaohui Feng, Wenwei Hu. Pontin, a new mutant p53 binding protein, promotes gain-of-function of mutant p53. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1221. doi:10.1158/1538-7445.AM2015-1221

Abstract 4446: Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis

Abstract Inhibition of de novo palmitate synthesis by fatty acid synthase (FASN) is a novel cancer therapeutic approach with strong biological rationale. Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. FASN expression increases with tumor progression in human tumors and associates with chemoresistance, metastasis, and diminished patient survival in many tumor types. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. TVB-2640 and TVB-3166 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences that exhibit anti-tumor activity in diverse preclinical tumor models. FASN inhibition induces tumor cell apoptosis by remodeling tumor cell membranes, blocking signal transduction, and reprogramming gene expression. These effects lead to inhibition of anchorage-independent tumor cell growth under lipid-rich conditions and inhibition of in vivo xenograft tumor growth in mice and rats. Studies to understand the mechanisms of action and biological consequences of FASN inhibition are guiding the discovery of tumors highly dependent on FASN activity and biomarkers for assessment of pharmacodynamic activity and patient selection. Using quantitative genomics and a variety of directed analytical approaches, we identified lipid, mRNA, and protein profiles in tumor cells that change in response to FASN inhibition. Subsets of the changes show correlation with FASN inhibitor sensitivity. Marker candidates, such as gene expression signatures that classify in vitro sensitivity to FASN inhibition, are being investigated for detection in clinical human tumor data sets. Our studies also identified FASN inhibitor-mediated mechanisms of action that function in specific tumor types and biomarkers with potential utility for selecting responsive patients and measuring tumor response. Inhibition of the Wnt/b-catenin pathway and expression of TCF-promoter-regulated genes such as c-Myc are examples. Tumors dependent on Wnt/b-catenin activity or with lower intracellular palmitate stores prior to drug treatment may be susceptible to FASN inhibitor treatment. Pairing MYC expression with additional markers improves the performance of MYC as a marker to select tumor cell lines highly sensitive to FASN inhibition. Biomarker candidates from in vitro studies have been examined in vivo using xenograft tumor studies. These preclinical data support 3-V Biosciences’ ongoing Phase I clinical study of a first-in-class FASN inhibitor TVB-2640, and help guide the next steps in its development. Citation Format: Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Glenn Hammonds, Marina Fridlib, Russell Johnson, Lily Hu, Allan Wagman, Marie O' Farrell, Douglas Buckley, George Kemble. Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4446. doi:10.1158/1538-7445.AM2015-4446

Pontin, a new mutant p53-binding protein, promotes gain-of-function of mutant p53.

Tumor-suppressor p53 is frequently mutated in human cancers. Many tumor-associated mutant p53 (mutp53) proteins gain new functions in promoting tumorigenesis, defined as gain-of-function (GOF). The mechanisms for mutp53 GOF are not well understood. Here, we report Pontin, a highly conserved AAA+ ATPase important for various cellular functions, as a new mutp53-binding protein. This Pontin-mutp53 interaction promotes mutp53 GOF in invasion, migration and anchorage-independent growth of tumor cells. The ATPase domain of Pontin is crucial for its promoting effect on mutp53 GOF; blocking the ATPase activity of Pontin by a Pontin-specific ATPase inhibitor or an ATPase-deficient dominant-negative Pontin expression vector greatly diminished mutp53 GOF. Pontin promotes mutp53 GOF through regulation of mutp53 transcriptional activity; knockdown of Pontin abolished the transcriptional regulation of mutp53 toward a group of genes. Furthermore, overexpression of Pontin in tumors is associated with the poor survival in cancer patients, especially those containing mutp53. Our results highlight an important role and mechanism for Pontin, a new mutp53 partner, in promoting mutp53 GOF in tumorigenesis.

Abstract 4521: P7170, a novel inhibitor of phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) and activin receptor-like kinase 1 (ALK1) shows anti-tumor activity in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) lacks ER, PR and Her2 receptor expression & accounts for nearly 10-15% of all breast cancer. It is an aggressive disease with high risk for early relapse & visceral metastasis leading to poor survival as compared to other subtypes. Currently, there is no targeted therapy beyond cytotoxic chemotherapy for TNBC. PI3K-mTOR pathway is often dysregulated (via mutations in PI3K or PTEN) in TNBC and hence offers an opportunity to develop a therapeutic to address the high unmet medical need in this disease. P7170, a small molecule mTOR/PI3K/ALK1 inhibitor with potent antitumor activity in various cancer types {Cancer Res, April 15,2012; 72(8 Supplement): 3742 and 3759} is currently undergoing Phase 1 clinical trial. Here, we report the evaluation of anti-tumor activity and the identification of potential pharmacodynamic markers of P7170 in a TNBC model. P7170 inhibited proliferation (IC50 range 5 - 38 nmol/L) in TNBC cell lines as determined using propidium iodide (PI) assay. In addition, P7170 inhibited anchorage-independent growth of tumor cells isolated from patient derived tumor xenografts evaluated in a clonogenic assay. PI3K-mTOR pathway proteins pS6 and p4EBP1 were potently inhibited whereas pAkt was modestly inhibited by P7170 in MDA-MB-231 cells as assessed by Western blot. It also inhibited migration of MDA-MB-231 cells in a transwell migration assay. P7170 at 1/3rd dose of Maximum tolerated dose (5 mg/kg, po) as a single agent resulted in 63% (P<0.05) tumor growth inhibition in MDA-MB-231 xenograft mouse model. Pharmacodynamic effects of P7170 were evaluated in the plasma, tumor and skin of MDA-MB-231 tumor bearing mice treated with P7170 (5 and 15 mg/kg) orally for 3 days. Expression of target genes was measured by qRT-PCR, tumor and skin proteins were measured by immunohistochemistry, and plasma proteins by ELISA. P7170 significantly reduced pS6 protein in a dose-and time-dependent manner in the tumor and skin. P7170 significantly reduced tumor p4EBP1 protein at the highest dose and reduced tumor CDC25A gene by 2-fold. In addition, P7170 dose-dependently reduced plasma IGF1/IGFBP3 ratios compared to vehicle group. Conclusion: These data suggest that P7170 demonstrates robust antiproliferative activities in vitro and anti-tumor activity in vivo in TNBC. Tumor pS6 protein, CDC25A transcript levels and skin pS6 levels were identified as pharmacodynamic markers of P7170 activity. P7170 could be a therapeutic option for TNBC patients, and warrants testing in a clinical trial. Citation Format: Veena R. Agarwal, Dimple Bhatia, Asavari Joshi, Prabha Mishra, Kalyani G. Bharadwaj, Aurelio S. lobo, Pranoy Menon, Payal Dhar, Prashant Pandey, Sreesha Srinivasa, vinay sonawane. P7170, a novel inhibitor of phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) and activin receptor-like kinase 1 (ALK1) shows anti-tumor activity in 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 4521. doi:10.1158/1538-7445.AM2014-4521

Abstract 3814: FAK inhibition targets nucleostemin, a nucleolar protein, impacting breast cancer spheroid growth and tumor progression

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that controls cell proliferation and survival downstream of integrin-matrix receptors. However, under deprivation of anchorage, FAK translocates to the nucleus. The nucleolus, a non-membrane structure within the nucleus, is important in the control of ribosome biogenesis, regulates the sequestration of nuclear proteins, and increased nucleolar size is a marker of aggressive tumors. Nucleostemin, a nucleolar-localized protein, acts to modulate cell cycle progression and anchorage-independent cell growth. Although many drugs have low efficacy on tumor cells cultured in suspension, nanomolar small molecule FAK inhibitor (PF-271 or PND-1186) addition can prevent anchorage-independent growth of tumor cells as spheroids through mechanism(s) that remain unresolved. Here, we show that pharmacological FAK inhibitor (FAK-I) treatment or kinase-dead (KD) FAK re-expression analyses reduced breast cancer (MDA-MB-231 and 4T1-L) anchorage-independent cell growth with a G0/G1 cell cycle block. This was associated with a specific decrease in nucleostemin protein levels. As FAK-KD forms a complex with nucleostemin, and proteasome inhibitor treatment increased nucleostemin levels in FAK-KD cells, FAK-KD may function as a scaffold to facilitate nucleostemin degradation. In orthotopic breast cancer mouse tumor models, oral FAK-I administration or FAK-KD expression resulted in smaller tumors, decreased Ki67 positive cells, and lower levels of tumor-associated nucleostemin. Ongoing experiments are focused on understanding the molecular mechanisms of FAK nucleolar localization and the regulation of nucleostemin protein levels. Our results point to a novel function of FAK in nucleolar function, spheroid growth, and breast cancer progression. Supported by NIH CA102310 and Susan G. Komen for the Cure (KG111237). Citation Format: Isabelle Tancioni, Sean Uryu, Nichol L. G. Miller, Christine Lawson, Christine Jean, Xiao Lei Chen, David D. Schlaepfer. FAK inhibition targets nucleostemin, a nucleolar protein, impacting breast cancer spheroid growth and tumor progression. [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 3814. doi:10.1158/1538-7445.AM2014-3814

Phosphatidylinositol Phosphate 5-Kinase Iγi2 in Association with Src Controls Anchorage-independent Growth of Tumor Cells

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.

MiR-21 coordinates tumor growth and modulates KRIT1 levels

miR-21 is overexpressed in tumors and it displays oncogenic activity. Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM). We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3′UTR and that this interaction is involved in tumor growth control. In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed. Our findings suggest that miR-21 promotes tumor cell growth, at least in part, by down-modulating the potential tumor suppressor KRIT1.

P38MAPK and Rho-dependent kinase are involved in anoikis induced by anicequol or 25-hydroxycholesterol in DLD-1 colon cancer cells

Anchorage-independent growth is evidence of the malignant transformation of cells. We previously reported the characterization of anicequol, a novel inhibitor of the anchorage-independent growth of tumor cells, and here we show that the effects of 25-hydroxycholesterol (25-HC) on colon cancer cells were very similar to those of anicequol. By analyzing the effects of inhibitors and performing RNA interference experiments, we found that p38 mitogen-activated protein kinase (p38MAPK) was involved in anicequol- and 25-HC-induced anoikis in DLD-1 cells. In addition, Rho-associated, coiled-coil containing protein kinase (ROCK) was also associated with anoikis induced by anicequol or 25-HC. Taken together, our findings suggest that activation of the p38MAPK and ROCK pathways might provide a new therapeutic strategy against cancer, and raise the possibility that tumor metastasis is influenced by 25-HC under physiological conditions.

Abstract 4142: Differential expression of PDCD4 in response to Resveratrol in breast cancer cells

Abstract Background: Programmed cell death 4 (PDCD4) is a key protein involved in protein synthesis, and thus, it controls cell growth and proliferation. Evidence indicates that PDCD4 is often downregulated in breast cancer and is associated with disease progression and metastasis. Resveratrol (RSV), a natural polyphenolic compound present in grapes and red wine, is a well known anti-oxidant capable of modulating various signaling pathways, leading to suppression of cell proliferation and increased apoptosis. However, it remains unclear regarding the effect of RSV on PDCD4. Therefore, in the present study, we investigated the effect of RSV on PDCD4 in human breast cancer MCF-7 and MDA-MB-231 cells. Methods: Cell growth was measured by MTT assays, and cell invasion was determined by matrigel chamber assays. Soft agar assays were used to determine anchorage independent growth. Protein levels from treated cells were analyzed by western blot. Real time RT-PCR was used to determine relative expression of genes. Phosphorylated Akt (pAkt) was also determined by immunohistochemistry in RSV-treated MDA-MB-231 cells. To determine whether microRNAs are involved in the RSV-mediated induction of PDCD4, we cloned PDCD4 3′-UTR into the downstream of the luciferase gene as a reporter. The effect of RSV on luciferase activity was tested in MCF-7 cells. PDCD4 was stably knocked down by RNAi in MCF-7 and MDA-MB-231 cells. Results: We showed that ectopic expression of PDCD4 suppressed anchorage independent tumor cell growth as well as invasion of MDA-MB-231 cells. By contrast, downregulation of PDCD4 by RNAi significantly increased cell invasion. Of interest, cell invasion was suppressed by RSV; at the same time, PDCD4 was induced significantly by RSV in MDA-MB-231 cells, which was associated with suppression of pAkt. Furthermore, we showed that both phosphorylated mTOR and S6K were suppressed by RSV in a dose dependent manner. Since PDCD4 is a downstream effector of Akt and mTOR, this may suggest that up-regulation by RSV is through the Akt-mTOR pathway in MDA-MB-231. On the other hand, PDCD4 was reduced by RSV in estrogen receptor-positive (ERα+) MCF-7 cells. To determine the mechanism of RSV-mediated PDCD4 expression in these two cell lines, we measured the levels of miR-21 which has been previously shown to directly target PDCD4. Strikingly, expression of miR-21 was also regulated differentially. For example, miR-21 was up-regulated in MCF-7 cells while it was slightly suppressed by RSV in MDA-MB-231 cells. In particular, we found that RSV suppressed PDCD4 by transcriptional regulation of miR-21 possibly through interacting with ERα in MCF-7 cells. Conclusion: Together, these results suggest that, as a tumor suppressor gene, PDCD4 expression is dependent on cellular contents in response to RSV, and thus, a better understanding of this RSV-mediated PDCD4 expression will provide new insight into the clinical implication of RSV for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4142. doi:1538-7445.AM2012-4142

Activation and Involvement of Ral GTPases in Colorectal Cancer

Current approaches to block KRAS oncogene function focus on inhibition of K-Ras downstream effector signaling. We evaluated the antitumor activity of selumetinib (AZD6244, ARRY-142886), a potent and selective MEK1/2 inhibitor, on a panel of colorectal carcinoma (CRC) cells and found no inhibition of KRAS mutant CRC cell anchorage-independent growth. Although AKT activity was elevated in KRAS mutant cells, and PI3K inhibition did impair the growth of MEK inhibitor-insensitive CRC cell lines, concurrent treatment with selumetinib did not provide additional antitumor activity. Therefore, we speculated that inhibition of the Ral guanine exchange factor (RalGEF) effector pathway may be a more effective approach for blocking CRC growth. RalGEFs are activators of the related RalA and RalB small GTPases and we found activation of both in CRC cell lines and patient tumors. Interfering RNA stable suppression of RalA expression reduced CRC tumor cell anchorage-independent growth, but surprisingly, stable suppression of RalB greatly enhanced soft agar colony size and formation frequency. Despite their opposing activities, both RalA and RalB regulation of anchorage-independent growth required interaction with RalBP1/RLIP76 and components of the exocyst complex. Interestingly, RalA interaction with the Exo84 but not Sec5 exocyst component was necessary for supporting anchorage-independent growth, whereas RalB interaction with Sec5 but not Exo84 was necessary for inhibition of anchorage-independent growth. We suggest that anti-RalA-selective therapies may provide an effective approach for KRAS mutant CRC.

Aberrant Overexpression of the Rgl2 Ral Small GTPase-specific Guanine Nucleotide Exchange Factor Promotes Pancreatic Cancer Growth through Ral-dependent and Ral-independent Mechanisms

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.

EGF-induced Grb7 Recruits and Promotes Ras Activity Essential for the Tumorigenicity of Sk-Br3 Breast Cancer Cells

Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.

EphA2 Mutation in Lung Squamous Cell Carcinoma Promotes Increased Cell Survival, Cell Invasion, Focal Adhesions, and Mammalian Target of Rapamycin Activation

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth

BackgroundApoptosis caused by inadequate or inappropriate cell-matrix interactions is defined as anoikis. Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood. We investigated the mechanisms of anoikis resistance of tumor cells.ResultsWe observed that cell aggregation in suspension promoted cell survival and proliferation. We demonstrated a correlation between tumor cell aggregation in suspension and cell growth in soft agar. Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates. We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2. Furthermore, RNA interference-mediated reduction of Pyk2 and PECAM-1 protein levels reduced cell aggregation and inhibited the growth of tumor cells in soft agar.ConclusionsThe data demonstrated that Pyk2 and PECAM-1 were critical mediators of both anchorage-independent growth and anoikis resistance in tumor cells.

Promotion of Anchorage-independent Growth by Cytoplasmic and Nuclear Histone Deacetylase 9

Genetic mutation is a trigger for the generation of malignant cells and an aberrant epigenetic status contributes to the maintenance of mutations and proliferation of mutated cells. Along with DNA methylation, histone modifications such as acetylation and methylation are significant to biological processes. Histone deacetylases (HDACs) are important epigenetic regulators of chromatin modifications and gene expression. Though several HDAC inhibitors are currently being tested in clinical trials, the roles of HDACs in malignant transformation remain unknown. Here, we showed that the expression of two forms of Hdac9, a full-length version (Hdac9FL) and a splicing variant lacking exon 7 (Hdac9Δ7), both class IIa HDACs, was up-regulated during chemically induced hepatocarcinogenesis. In addition, we found that HDAC9FL and HDAC9Δ7 are located in the nucleus and cytoplasm, respectively. We also found their nuclear localization and nuclear export signals to be encoded in exon 7 and exon 25, respectively. Though the two isoforms could not transform mouse NIH-3T3 fibroblasts, they promoted tumor cell anchorage-independent growth on soft agarose. The HDAC9 variants do not seem to cause cell transformation, but cytoplasmic and nuclear HDAC9 may contribute to the survival of malignant cells in the early stages of hepatocarcinogenesis.

A Selective Phosphatase of Regenerating Liver Phosphatase Inhibitor Suppresses Tumor Cell Anchorage-Independent Growth by a Novel Mechanism Involving p130Cas Cleavage

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.

Osteopontin as a target for cancer therapy

Osteopontin (OPN) is a glycophosphoprotein cytokine that has multiple functions. OPN is expressed and secreted by various cells, and has a role in cell adhesion, chemotaxis, prevention of apoptosis, invasion, migration and anchorage-independent growth of tumor cells. Extensive research has demonstrated the pivotal participation of OPN in the regulation of cell signaling which controls neoplastic and malignant transformation. The elevated expression of OPN has been observed in a variety of cancers. OPN has been linked with tumor metastasis and signifies a poor prognosis for the patient. This review details the mechanisms by which OPN facilitates these pathological events. It will also show that gaining an understanding of the mechanism of OPN's action at a cellular level has led to the development of a number of therapeutic strategies against the cytokine. These include inhibiting its expression, antagonizing cell surface receptor activation and blocking downstream cell signaling pathways. In addition to the potential of these therapies, serum levels of OPN could be used as a diagnostic and prognostic marker. The authors propose that with further research and development, osteopontin directed treatment could greatly enhance outcomes for cancer patients.