Cross‐signaling among phosphinositide‐3 kinase,mitogen‐activated protein kinase and sonic hedgehog pathways exists in esophageal cancer

The hedgehog (Hh) signaling pathway is essential for the development of tissues and organs. Hyperactive Hh signaling has been implicated in many gastric cancers, including esophageal cancer. However, the interaction between the Hh pathway and other potential signaling pathways in primary esophageal tumorigenesis has not been well investigated. In our study, we found that esophageal cancer cells expressed Hh signaling molecules and that the hyperexpression of Hh target genes was related to protein kinase B (AKT) activation but not extracellular signal‐regulated kinase activation. We analyzed the relationship between Gli1 or p‐AKT expression and clinicopathological features in esophageal carcinoma samples and found that Gli1 expression was associated with lymph vessel invasion (p = 0.016), blood vessel invasion (p = 0.006) and a poor prognosis (p = 0.003), and p‐AKT expression was associated with blood vessel invasion (p = 0.031) and a poor prognosis (p = 0.031). We also studied the relationship between Hh and phosphinositide‐3 kinase (PI3K)/AKT or mitogen‐activated protein kinase (MAPK) signaling pathways in both TE‐1 and TE‐10 cell lines. We found that the PI3K/AKT pathway played a critical role in Hh signaling after stimulation with epidermal growth factor, Gβγ and N‐Shh. Conversely, PI3K/AKT and MAPK signaling cooperated with the Shh pathway to promote esophageal cancer cell survival and proliferation. The results from esophageal cancer cells shed light on the significance of Hh signaling in esophageal tumor formation and the crosstalk of the Hh pathway with other basic signaling pathways, which is consistent with that observed in human tumor samples.     

Human epidermal growth factor receptor 2 regulates angiopoietin-2 expression in breast cancer via AKT and mitogen-activated protein kinase pathways

Abnormal activation of human epidermal growth factor receptor 2 (HER2; ErbB-2) in breast tumors results in increased metastasis and angiogenesis, as well as reduced survival. Here, we show that angiopoietin-2 (Ang-2) expression correlates with HER2 activity in human breast cancer cell lines. Inhibiting HER2 activity with anti-HER2 monoclonal antibody trastuzumab (Herceptin) or HER2 short interfering RNA in tumor cells down-regulates Ang-2 expression. Consistent with the important roles of AKT and mitogen-activated protein kinase in the HER2 signaling pathway, AKT and ERK mitogen-activated protein kinase (MAPK) kinase activity is necessary for Ang-2 up-regulation by HER2. Moreover, overexpression of HER2 protein up-regulates Ang-2 expression. Heregulin-beta1-induced Ang-2 up-regulation is abrogated when AKT and ERK kinase activity are blocked. Immunohistochemical analysis of HER2 and Ang-2 proteins in human breast carcinomas shows that Ang-2 expression in breast cancer correlates with HER2 expression. These studies provide evidence that the Ang-2 gene is regulated by HER2 activity in breast cancer, and propose an additional mechanism for HER2 contributing to tumor angiogenesis and metastasis.     

Targeting the RNA splicing machinery as a novel treatment strategy for pancreatic carcinoma

Aberrant patterns of pre-mRNA splicing have been established for many human malignancies, yet the mechanisms responsible for these tumor-specific changes remain undefined and represent a promising area for therapeutic intervention. Using immunohistochemistry, we have localized the expression of a central splicing regulator, serine-arginine protein kinase 1 (SRPK1), to the ductular epithelial cells within human pancreas and have further shown its increased expression in tumors of the pancreas, breast, and colon. Small interfering RNA-mediated down-regulation of SRPK1 in pancreatic tumor cell lines resulted in a dose-dependent decrease in proliferative capacity and increase in apoptotic potential. Coordinately, the disruption of SRPK1 expression resulted in enhanced sensitivity of tumor cells to killing by gemcitabine and/or cisplatin. A dose-dependent reduction in the phosphorylation status of specific SR proteins was detected following the down-regulation of SRPK1 and is likely responsible for the observed alterations in expression of proteins associated with apoptosis and multidrug resistance. These data support SRPK1 as a new, potential target for the treatment of pancreatic ductular cancer that at present remains largely unresponsive to conventional therapies. Furthermore, these results support the development of innovative therapies that target not only specific splice variants arising during tumorigenesis but also the splice regulatory machinery that itself may be abnormal in malignant cells.     

Enhanced apoptosis and tumor growth suppression elicited by combination of MEK (selumetinib) and mTOR kinase inhibitors (AZD8055)

The mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT signaling pathways interact at multiple nodes in cancer, including at mTOR complexes, suggesting an increased likelihood of redundancy and innate resistance to any therapeutic effects of single pathway inhibition. In this study, we investigated the therapeutic effects of combining the MAPK extracellular signal-regulated kinase (MEK)1/2 inhibitor selumetinib (AZD6244) with the dual mTORC1 and mTORC2 inhibitor (AZD8055). Concurrent dosing in nude mouse xenograft models of human lung adenocarcinoma (non-small cell lung cancers) and colorectal carcinoma was well tolerated and produced increased antitumor efficacy relative to the respective monotherapies. Pharmacodynamic analysis documented reciprocal pathway inhibition associated with increased apoptosis and Bim expression in tumor tissue from the combination group, where key genes such as DUSP6 that are under MEK functional control were also modulated. Our work offers a strong rationale to combine selumetinib and AZD8055 in clinical trials as an attractive therapeutic strategy.     

American ginseng (Panax quinquefolius L.) extract alters mitogen-activated protein kinase cell signaling and inhibits proliferation of MCF-7 cells

Ginseng has been shown to inhibit cancer cell proliferation and tumor growth, however the mechanisms underlying this inhibition have yet to be elucidated. An inhibitory effect of hot water-extracted American ginseng (Panax quinquefolius L.) root on cell proliferation was demonstrated using MCF-7 human breast cancer cells treated with a wide concentration range of the ginseng extract (GE) for 6 days. The effects of GE were concentration-dependent with an IC50 of 0.49 microg/microl and the minimum exposure time to elicit an inhibitory response was 24 hours. Using an antibody microarray, it was determined that several key cell survival proteins were altered in GE-treated cells, including several members of the mitogen-activated protein kinase (MAPK) family. A GE-induced decrease in phospho-MEK1/2 and -ERK1/2 and an increase in phospho-Raf-1 were observed and verified using Western blot analysis. Furthermore, mRNA and protein expression of the Raf-1 kinase inhibitor protein (RKIP) was shown to be transiently, yet significantly, upregulated following GE treatment. These results suggest that American ginseng may act to inhibit breast cancer cell proliferation by increasing the expression of RKIP, resulting in inhibition of the MAPK pathway. This novel mechanism has implications in the potential prevention and treatment of breast cancer.     

The MMAC1 tumor suppressor phosphatase inhibits phospholipase C and integrin-linked kinase activity

Loss of the tumor suppressor MMAC1 has been shown to be involved in breast, prostate and brain cancer. Consistent with its identification as a tumor suppressor, expression of MMAC1 has been demonstrated to reduce cell proliferation, tumorigenicity, and motility as well as affect cell-cell and cell-matrix interactions of malignant human glioma cells. Subsequently, MMAC1 was shown to have lipid phosphatase activity towards PIP3 and protein phosphatase activity against focal adhesion kinase (FAK). The lipid phosphatase activity of MMAC1 results in decreased activation of the PIP3-dependent, anti-apoptotic kinase, AKT. It is thought that this inhibition of AKT culminates with reduced glioma cell proliferation. In contrast, MMAC1's effects on cell motility, cell - cell and cell - matrix interactions are thought to be due to its protein phosphatase activity towards FAK. However, recent studies suggest that the lipid phosphatase activity of MMAC1 correlates with its ability to be a tumor suppressor. The high rate of mutation of MMAC1 in late stage metastatic tumors suggests that effects of MMAC1 on motility, cell - cell and cell - matrix interactions are due to its tumor suppressor activity. Therefore the lipid phosphatase activity of MMAC1 may affect PIP3 dependent signaling pathways and result in reduced motility and altered cell - cell and cell - matrix interactions. We demonstrate here that expression of MMAC1 in human glioma cells reduced intracellular levels of inositol trisphosphate and inhibited extracellular Ca2+ influx, suggesting that MMAC1 affects the phospholipase C signaling pathway. In addition, we show that MMAC1 expression inhibits integrin-linked kinase activity. Furthermore, we show that these effects require the catalytic activity of MMAC1. Our data thus provide a link of MMAC1 to PIP3 dependent signaling pathways that regulate cell - matrix and cell - cell interactions as well as motility. Lastly, we demonstrate that AKT3, an isoform of AKT highly expressed in the brain, is also a target for MMAC1 repression. These data suggest an important role for AKT3 in glioblastoma multiforme. We therefore propose that repression of multiple PIP3 dependent signaling pathways may be required for MMAC1 to act as a tumor suppressor.     

Molecular cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines

hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2(+)/ER(-)/Ki67(+) unfavorable prognostic phenotype. In vitro, neuregulin-1 up-regulates whereas Herceptin treatment down-modulates hMena expression, suggesting that it may couple tyrosine kinase receptor signaling to the actin cytoskeleton. Herein, we report the cloning of hMena and of a splice variant, hMena(+11a), which contains an additional exon corresponding to 21 amino acids located in the EVH2 domain, from a breast carcinoma cell line of epithelial phenotype. Whereas hMena overexpression consistently characterizes the transformed phenotype of tumor cells of different lineages, hMena(+11a) isoform is concomitantly present only in epithelial tumor cell lines. In breast cancer cell lines, epidermal growth factor (EGF) treatment promotes concomitant up-regulation of hMena and hMena(+11a), resulting in an increase of the fraction of phosphorylated hMena(+11a) isoform only. hMena(+11a) overexpression and phosphorylation leads to increased p42/44 mitogen-activated protein kinase (MAPK) activation and cell proliferation as evidenced in hMena(+11a)-transfected breast cancer cell lines. On the contrary, hMena knockdown induces reduction of p42/44 MAPK phosphorylation and of the proliferative response to EGF. The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer.     

Resveratrol-induced growth inhibition in MDA-MB-231 breast cancer cells is associated with mitogen-activated protein kinase signaling and protein translation.

Resveratrol (3,4',5-trans-trihydroxystilbene) is a natural compound found in grapes and several medicinal plants and has been shown to have anticancer effects on various human cancer cells. The aim of this study was to further investigate the molecular mechanism of this anticancer effect. Resveratrol effect on cell growth, morphology and gene expression was investigated in estrogen receptor-negative MDA-MB-231 human breast cancer cell line. We show here that resveratrol-induced growth inhibition in the estrogen receptor negative MDA-MB-231 breast cancer cells is due to the induction of apoptosis as demonstrated by morphological, nuclear staining and PARP cleavage analysis. Resveratrol-induced growth inhibition was associated with transient activation of p44/42 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204). Most importantly, resveratrol inhibited both the phosphorylation at Ser240/244 and the expression of the pS6 ribosomal protein. This protein is known to play an important role in the translation of mRNAs that have oligopyrimidine tracts in their 5' untranslated regions. Interestingly, only MAPK inhibitor was able to block resveratrol-induced growth inhibition suggesting that effects of resveratrol on cell growth are dependent on MAPK signaling. The data demonstrated that resveratrol-induced apoptosis is associated with MAPK signaling and with the inhibition of proteins that are involved in protein translation. This is the first data linking resveratrol with downregulation of protein translation via p44/42 MAPK and S6 ribosomal protein. We propose to use these proteins as predictive biomarkers to evaluate the treatment efficacy of resveratrol in estrogen receptor-negative human breast cancer.     

Mutations in BRAF and KRAS converge on activation of the mitogen-activated protein kinase pathway in lung cancer mouse models

Mutations in the BRAF and KRAS genes occur in approximately 1% to 2% and 20% to 30% of non-small-cell lung cancer patients, respectively, suggesting that the mitogen-activated protein kinase (MAPK) pathway is preferentially activated in lung cancers. Here, we show that lung-specific expression of the BRAF V600E mutant induces the activation of extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway and the development of lung adenocarcinoma with bronchioloalveolar carcinoma features in vivo. Deinduction of transgene expression led to dramatic tumor regression, paralleled by dramatic dephosphorylation of ERK1/2, implying a dependency of BRAF-mutant lung tumors on the MAPK pathway. Accordingly, in vivo pharmacologic inhibition of MAPK/ERK kinase (MEK; MAPKK) using a specific MEK inhibitor, CI-1040, induced tumor regression associated with inhibition of cell proliferation and induction of apoptosis in these de novo lung tumors. CI-1040 treatment also led to dramatic tumor shrinkage in murine lung tumors driven by a mutant KRas allele. Thus, somatic mutations in different signaling intermediates of the same pathway induce exquisite dependency on a shared downstream effector. These results unveil a potential common vulnerability of BRAF and KRas mutant lung tumors that potentially affects rational deployment of MEK targeted therapies to non-small-cell lung cancer patients.     

Cell growth inhibition by all-trans retinoic acid in SKBR-3 breast cancer cells: involvement of protein kinase Calpha and extracellular signal-regulated kinase mitogen-activated protein kinase

All-trans retinoic acid (ATRA), a synthetic derivative of vitamin A, inhibits the growth of breast cancer cells. To elucidate the mechanism by which ATRA causes cell growth inhibition, we examined changes in cell cycle and intracellular signaling pathways, focusing on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Using the estrogen receptor-negative, retinoid receptor-positive breast cancer cell line SKRB-3, we found that treatment with ATRA significantly decreased the expression of PKCalpha, as well as reducing ERK MAPK phosphorylation. ATRA treatment leads to dephosphorylation of Rb, and consequently to G(1) arrest. Marked changes in the expression of cyclins (particularly cyclins A and E) were observed in SKBR-3 cells treated with ATRA. Using a series of pharmacological and molecular approaches, we found evidence that ATRA-induced SKBR-3 cell growth inhibition involves the deregulation of the PKCalpha-MAPK pathway. These data suggest that retinoids interfered with signal transduction pathways that are crucial for cell cycle progression, and highlight the complexities of the biological effects of retinoid derivatives.     

Activation of mitogen-activated protein kinase in estrogen receptor alpha-positive breast cancer cells in vitro induces an in vivo molecular phenotype of estrogen receptor alpha-negative human breast tumors

Breast cancer presents as either estrogen receptor alpha (ERalpha) positive or negative, with ERalpha+ tumors responding to antiestrogen therapy and having a better prognosis. By themselves, mRNA expression signatures of estrogen regulation in ERalpha+ breast cancer cells do not account for the vast molecular differences observed between ERalpha+ and ERalpha- cancers. In ERalpha- tumors, overexpression of epidermal growth factor receptor (EGFR) or c-erbB-2, leading to increased growth factor signaling, is observed such that mitogen-activated protein (MAP) kinase (MAPK) is significantly hyperactivated compared with ERalpha+ breast cancer. In ERalpha+/progesterone receptor-positive, estrogen-dependent MCF-7 breast cancer cells, we stably overexpressed EGFR or constitutively active erbB-2, Raf, or MAP/extracellular signal-regulated kinase kinase, resulting in cell lines exhibiting hyperactivation of MAPK, estrogen-independent growth, and the reversible down-regulation of ERalpha expression. By global mRNA profiling, we found a "MAPK signature" of approximately 400 genes consistently up-regulated or down-regulated in each of the MAPK+ cell lines. In several independent profile data sets of human breast tumors, the in vitro MAPK signature was able to accurately distinguish ER+ from ER- tumors. In addition, our in vitro mRNA profile data revealed distinct mRNA signatures specific to either erbB-2 or EGFR activation. A subset of breast tumor profiles was found to share extensive similarities with either the erbB-2-specific or the EGFR-specific signatures. Our results confirm that increased MAPK activation causes loss of ERalpha expression and suggest that hyperactivation of MAPK plays a role in the generation of the ERalpha- phenotype in breast cancer. These MAPK+ cell lines are excellent models for investigating the underlying mechanisms behind the ERalpha- phenotype.     

Ibuprofen disrupts a WNK1/GSK3β/SRPK1 protein complex required for expression of tumor-related splicing variant RAC1B in colorectal cells

A major risk factor promoting tumor development is chronic inflammation and the use of nonsteroidal anti-inflammatory drugs (NSAID), including ibuprofen, can decrease the risk of developing various types of cancer, including colorectal cancer (CRC). Although the molecular mechanism behind the antitumor properties of NSAIDs has been largely attributed to inhibition of cyclooxygenases (COXs), several studies have shown that the chemopreventive properties of ibuprofen also involve multiple COX-independent effects. One example is its ability to inhibit the alternative splicing event generating RAC1B, which is overexpressed in a specific subset of BRAF-mutated colorectal tumors and sustains cell survival. Here we describe the mechanism by which ibuprofen prevents RAC1B alternative splicing in a BRAF mutant CRC cell line: it leads to decreased translocation of SRPK1 and SRSF1 to the nucleus and is regulated by a WNK1/GSK3β/SRPK1 protein kinase complex. Surprisingly, we demonstrate that ibuprofen does not inhibit the activity of any of the involved kinases but rather promotes disassembly of this regulatory complex, exposing GSK3β serine 9 to inhibitory phosphorylation, namely by AKT, which results in nuclear exclusion of SRPK1 and SRSF1 hypophosphorylation. The data shed new light on the biochemical mechanisms behind ibuprofen’s action on alternative spliced RAC1B and may support its use in personalized approaches to CRC therapy or chemoprevention regimens.     

Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors

Elevated focal adhesion kinase (FAK) expression occurs in advanced cancers, yet a signaling role for FAK in tumor progression remains undefined. Here, we suppressed FAK activity in 4T1 breast carcinoma cells resulting in reduced FAK Y925 phosphorylation, Grb2 adaptor protein binding to FAK, and signaling to mitogen-activated protein (MAP) kinase (MAPK). Loss of a FAK-Grb2-MAPK linkage did not affect 4T1 cell proliferation or survival in culture, yet FAK inhibition reduced vascular endothelial growth factor (VEGF) expression and resulted in small avascular tumors in mice. This FAK-Grb2-MAPK linkage was essential in promoting angiogenesis as reconstitution experiments using Src-transformed FAK-null fibroblasts revealed that point mutations affecting FAK catalytic activity (R454) or Y925 phosphorylation (F925) disrupted the ability of FAK to promote MAPK- and VEGF-associated tumor growth. Notably, in both FAK-inhibited 4T1 and Src-transformed FAK-null cells, constitutively activated (CA) mitogen-activated protein kinase kinase 1 (MEK1) restored VEGF production and CA-MEK1 or added VEGF rescued tumor growth and angiogenesis. These studies provide the first biological support for Y925 FAK phosphorylation and define a novel role for FAK activity in promoting a MAPK-associated angiogenic switch during tumor progression.     

Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.     

Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer

Mitogen-activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival and differentiation. Aberrant regulation of MAPK cascades contribute to cancer and other human diseases. In particular, the extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intense research scrutiny leading to the development of pharmacologic inhibitors for the treatment of cancer. ERK is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine/threonine kinases. Raf activates the MAPK/ERK kinase (MEK)1/2 dual-specificity protein kinases, which then activate ERK1/2. The mutational activation of Raf in human cancers supports the important role of this pathway in human oncogenesis. Additionally, the Raf-MEK-ERK pathway is a key downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers. Finally, Ras is a key downstream effector of the epidermal growth factor receptor (EGFR), which is mutationally activated and/or overexpressed in a wide variety of human cancers. ERK activation also promotes upregulated expression of EGFR ligands, promoting an autocrine growth loop critical for tumor growth. Thus, the EGFR-Ras-Raf-MEK-ERK signaling network has been the subject of intense research and pharmaceutical scrutiny to identify novel target-based approaches for cancer treatment. In this review, we summarize the current status of the different approaches and targets that are under evaluation and development for the therapeutic intervention of this key signaling pathway in human disease.     

Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis

Although mutated forms of ras are not associated with the majority of breast cancers (<5%), there is considerable experimental evidence that hyperactive Ras can promote breast cancer growth and development. Therefore, we determined whether Ras and Ras-responsive signaling pathways were activated persistently in nine widely studied human breast cancer cell lines. Although only two of the lines harbor mutationally activated ras, we found that five of nine breast cancer cell lines showed elevated active Ras-GTP levels that may be due, in part, to HER2 activation. Unexpectedly, activation of two key Ras effector pathways, the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT signaling pathways, was not always associated with Ras activation. Ras activation also did not correlate with invasion or the expression of proteins associated with tumor cell invasion (estrogen receptor alpha and cyclooxygenase 2). We then examined the role of Ras signaling in mediating resistance to matrix deprivation-induced apoptosis (anoikis). Surprisingly, we found that ERK and phosphatidylinositol 3'-kinase/AKT activation did not have significant roles in conferring anoikis resistance. Taken together, these observations show that Ras signaling exhibits significant cell context variations and that other effector pathways may be important for Ras-mediated oncogenesis, as well as for anoikis resistance, in breast cancer. Additionally, because ERK and AKT activation are not strictly associated with Ras activation, pharmacological inhibitors of these two signaling pathways may not be the best approach for inhibition of aberrant Ras function in breast cancer treatment.