A novel NHE1-centered signaling cassette drives epidermal growth factor receptor-dependent pancreatic tumor metastasis and is a target for combination therapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers principally because of early invasion and metastasis. The epidermal growth factor receptor (EGFR) is essential for PDAC development even in the presence of Kras, but its inhibition with erlotinib gives only a modest clinical response, making the discovery of novel EGFR targets of critical interest. Here, we revealed by mining a human pancreatic gene expression database that the metastasis promoter Na(+)/H(+) exchanger (NHE1) associates with the EGFR in PDAC. In human PDAC cell lines, we confirmed that NHE1 drives both basal and EGF-stimulated three-dimensional growth and early invasion via invadopodial extracellular matrix digestion. EGF promoted the complexing of EGFR with NHE1 via the scaffolding protein Na+/H+ exchanger regulatory factor 1, engaging EGFR in a negative transregulatory loop that controls the extent and duration of EGFR oncogenic signaling and stimulates NHE1. The specificity of NHE1 for growth or invasion depends on the segregation of the transient EGFR/Na+/H+ exchanger regulatory factor 1/NHE1 signaling complex into dimeric subcomplexes in different lipid raftlike membrane domains. This signaling complex was also found in tumors developed in orthotopic mice. Importantly, the specific NHE1 inhibitor cariporide reduced both three-dimensional growth and invasion independently of PDAC subtype and synergistically sensitized these behaviors to low doses of erlotinib.

The scaffolding protein NHERF1 sensitizes EGFR-dependent tumor growth, motility and invadopodia function to gefitinib treatment in breast cancer cells.

Triple negative breast cancer (TNBC) patients cannot be treated with endocrine therapy or targeted therapies due to lack of related receptors. These patients overexpress the epidermal growth factor receptor (EGFR), but are resistant to tyrosine kinase inhibitors (TKIs) and anti-EGFR therapies. Mechanisms suggested for resistance to TKIs include EGFR independence, mutations and alterations in EGFR and in its downstream signalling pathways. Ligand-induced endocytosis and degradation of EGFR play important roles in the downregulation of the EGFR signal suggesting that its activity could be regulated by targeting its trafficking. Evidence in normal cells showing that the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF1) can associate with EGFR to regulate its trafficking, led us to hypothesize that NHERF1 expression levels could regulate EGFR trafficking and functional expression in TNBC cells and, in this way, modulate its role in progression and response to treatment. We investigated the subcellular localization of NHERF1 and its interaction with EGFR in a metastatic basal like TNBC cell model, MDA-MB­231, and the role of forced NHERF1 overexpression and/or stimulation with EGF on the sensitivity to EGFR specific TKI treatment with gefitinib. Stimulation with EGF induces an interaction of NHERF1 with EGFR to regulate its localization, degradation and function. NHERF1 overexpression is sufficient to drive its interaction with EGFR in non-stimulated conditions, inhibits EGFR degradation and increases its retention time in the plasma membrane. Importantly, NHERF1 overexpression strongly sensitized the cell to the pharmacological inhibition by gefitinib of EGFR-driven growth, motility and invadopodia-dependent ECM proteolysis. The further determination of how the NHERF1­EGFR interaction is regulated may improve our understanding of TNBC resistance to the action of existing anticancer drugs.

The presence of clustered circulating tumor cells (CTCs) and circulating cytokines define an aggressive phenotype in metastatic colorectal cancer.

PURPOSE: Colon carcinoma is a malignant tumor showing a marked preference to metastasize to distant organs. The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. However, whether circulating cytokines are linked to the circulation of tumor cells, as individual cells or clusters, remain unclear. In this study, we investigated the circulating levels of TGF-beta, CXCL1, VEGF and PAI-1 as potential bioindicators of the presence of CTCs in patients with metastatic colon cancer. METHODS: Circulating tumor cells (CTCs) were isolated from peripheral blood by immunomagnetic separation and phenotypically characterized in a cohort of 103 patients with metastatic colon cancer. TGF-beta, CXCL1, VEGF and PAI-1 concentrations were determined by immunoassay in plasma samples from the same patients. RESULTS: We detected two different populations of CTCs, single cells or clusters in patients with metastatic colon cancer. Importantly, we found that the presence of clustered CTCs is significantly associated with elevated circulating levels of TGF-beta and CXCL1 and with reduced overall survival. Finally, we observed that circulating levels of cytokines are differently associated with the two populations of CTCs. CONCLUSIONS: Taken together, these findings show that detection of clustered CTCs represents a negative prognostic factor in patients with metastatic colon cancer. The presence of clustered CTCs is associated with elevated circulating levels of cytokines such as TGF-beta and CXCL1. This suggests an additional role for circulating cytokines as predictive tool for cancer prognosis and diagnosis of minimal residual disease as well as assessment of tumor sensitivity to anticancer therapy.

Clinical and prognostic role of circulating MMP-2 and its inhibitor TIMP-2 in HCC patients prior to and after trans-hepatic arterial chemo-embolization.

BACKGROUND AND AIMS: Trans-hepatic arterial chemo-embolization is the most commonly used treatment for unresectable hepatocellular carcinoma. The prognostic impact of tumor biomarkers has not therefore been evaluated in this treatment. Imbalance between matrix metalloproteinase-2 and tissue inhibitor metalloproteinase-2 is considered to play an important role in extracellular matrix remodeling and degradation. Higher serum levels of MMP-2 have been shown to predict a poor prognosis and shorter overall survival in HCC after TACE. The objective of this study was to evaluate the serum levels of MMP-2 and TIMP-2 in HCC patients before and after TACE to evaluate their clinical significance and usefulness as prognostic biomarkers. METHODS: MMP-2 and TIMP-2 levels were measured by ELISA in 75 HCC patients and 30 healthy controls. Sera MMP-2 and TIMP-2 were correlated with clinico-pathological features. RESULTS: The mean serum MMP-2 and TIMP-2 levels of HCC patients before TACE were 1700±71ng/mL and 89±45ng/mL respectively, significantly higher than that of the control group: 771±60ng/mL (p<0.0001, t-test) and 25.7±20ng/mL respectively (p<0.0001, t-test). A significant decrease of MMP-2 levels after 1 and 3months compared to baseline time was observed (p<0.0001), while with TIMP-2 a gradual increase in serum before and after TACE (p<0.01) was detected. No significant correlation between serum MMP-2 levels and other clinico-pathological features was observed. Patients with serum MMP-2 >1500ng/mL (median value) had worse overall and recurrence-free survival compared with those with serum MMP-2 levels <1500ng/mL before treatment. CONCLUSION: Higher serum MMP-2 levels and MMP-2/TIMP-2 ratio could predict poor prognosis after TACE, suggesting prognostic role of these biomarkers in HCC.

Circulating levels of transforming growth factor-ßeta (TGF-ß) and chemokine (C-X-C motif) ligand-1 (CXCL1) as predictors of distant seeding of circulating tumor cells in patients with metastatic breast cancer.

BACKGROUND: The presence of circulating tumor cells (CTCs) in the peripheral blood is a prerequisite for the formation of distant metastases. Transforming growth factor-ßeta (TGF-ß) and Chemokine (C-X-C Motif) Ligand-1 (CXCL1) are cytokines involved in the colonization of distant sites by CTCs in several pre-clinical animal models. However, their role is poorly-investigated in patients with metastatic cancer. Here, we investigated whether circulating levels of TGF-ß and CXCL1 are predictors of CTC seeding in preferential distant sites in patients with metastatic breast cancer. MATERIALS AND METHODS: CTCs were isolated from the peripheral blood of 61 patients with metastatic breast cancer by immunomagnetic separation. Plasma samples were collected from the same patients and assayed for TGF-ß and CXCL1 by enzyme-linked immunoassay. RESULTS: Patients were grouped in CK1+/- (N<10), CK2+ (N ≥ 10<50) and CK3+ (N ≥ 50), according to the number (N) of cytokeratin 7/8-positive CTCs: the highest number of CK7/8-positive CTCs was detected in patients with negative Human epidermal growth factor receptor-2 (HER-2/NEU) status (p<0.0001) antigen, identified by the monoclonal antibody Ki-67 (Ki-67) ≥ 15% (p=0.003), Carcinoma antigen 15-3 (CA-15.3) ≥ 40 U/ml (p=0.004) and those with lung metastases (p=0.01). We found that elevated plasma concentrations of TGF-ß and CXCL1 are predictive for the detection of CTCs. In particular, patients with CK3+ CTCs and plasma concentrations of TGF-ß and CXCL1 higher than the median value had a poor prognosis in comparison to patients with CK1+/- CTCs and TGF-ß and CXCL1 concentrations below the median value. CONCLUSION: Our study shows that elevated circulating levels of TGF-ß and CXCL1 are associated with a poor prognosis, and higher detection of CTCs and propensity of these cells to seed lung metastases in patients with breast cancer.

Co-expression of CD133(+)/CD44(+) in human colon cancer and liver metastasis.

Although relatively good therapeutic results are achieved in non-advanced cancer, the prognosis of the advanced colon cancer still remains poor, dependent on local or distant recurrence of the disease. One of the factors responsible for recurrence is supposed to be cancer stem cells (CSCs) or tumor-initiating cells, which are a population of cancer cells with ability to perpetuate themselves through self-renewal and to generate differentiated cells, thought to be responsible for tumor recurrence. This study globally approach the possible role of tissue-derived stem cells in the initiation of colon cancer and its metastatic process in the liver. Fresh surgical specimens from colon cancer, non-tumor tissue and liver metastasis were obtained directly from the operating room, examined, and immediately processed. CSCs were selected under serum-free conditions and characterized by CD44 and CD133 expression levels. CD133(+)/CD44(+) cell populations were then investigated in paraffin-embedded tissues and circulating tumor cells isolated from peripheral blood of the same group of colon cancer patients. Our data demonstrate that metastatic properties of cell populations from blood and liver metastasis, differently from primitive tumors, seem to be strictly related to the phenotype CD133 positive and CD44 positive.

Involvement of nuclear NHERF1 in colorectal cancer progression.

NHERF1 (Na+/H+ exchanger regulatory factor 1) is expressed in the luminal membrane of many epithelia, and associated with proteins involved in tumor progression. Alterations of NHERF1 expression in different sites of metastatic colorectal cancer (mCRC) suggest a dynamic role of this protein in colon carcinogenesis. We focused on the observation of the altered expression of NHERF1 from non-neoplastic tissues to metastatic sites by immunohistochemistry. Moreover, we studied, by immunofluorescence, the colocalization between NHERF1 and the epidermal growth factor receptor (EGFR), whose overexpression is implicated in CRC progression. NHERF1 showed a different localization and expression in the examined sites. The distant non-neoplastic tissues showed NHERF1 mostly expressed at the apical membrane, while in surrounding non-neoplastic tissue decreased the apical membrane and increased cytoplasmic immunoreactivity. In adenomas a shift from apical membrane to cytoplasmic localization and nuclear expression were observed. Cytoplasmic staining in the tumor, and metastatic sites was stronger than surrounding non-neoplastic tissue. Furthermore, nuclear NHERF1 expression was noted in 80% of all samples and surprisingly, it appeared already in adenoma lesions, suggesting that NHERF1 represents an early marker of pre-morphological triggering of colorectal carcinogenesis. Then, in few tumors a positive direct correlation between membrane NHERF1 and EGFR expression was evidenced by their colocalization. Nuclear NHERF1 expression, present in the early stages of carcinogenesis and related with poor prognosis, may contribute to the onset of malignant phenotype. Specifically, we hypothesize the direct involvement of nuclear NHERF1 in both carcinogenesis and progression and its role as a potential colorectal cancer marker.

NHERF1 acts as a molecular switch to program metastatic behavior and organotropism via its PDZ domains.

Metastatic cells are highly plastic for differential expression of tumor phenotype hallmarks and metastatic organotropism. The signaling proteins orchestrating the shift of one cell phenotype and organ pattern to another are little known. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a molecular pathway organizer, PDZ-domain protein that recruits membrane, cytoplasmic, and cytoskeletal signaling proteins into functional complexes. To gain insight into the role of NHERF1 in metastatic progression, we stably transfected a metastatic breast cell line, MDA-MB-231, with an empty vector, with wild-type NHERF1, or with NHERF1 mutated in either the PDZ1- or PDZ2-binding domains to block their binding activities. We observed that NHERF1 differentially regulates the expression of two phenotypic programs through its PDZ domains, and these programs form the mechanistic basis for metastatic organotropism. The PDZ2 domain promotes visceral metastases via increased invadopodia-dependent invasion and anchorage-independent growth, as well as by inhibition of apoptosis, whereas the PDZ1 domain promotes bone metastases by stimulating podosome nucleation, motility, neoangiogenesis, vasculogenic mimicry, and osteoclastogenesis in the absence of increased growth or invasion. Collectively, these findings identify NHERF1 as an important signaling nexus for coordinating cell structure with metastatic behavior and identifies the "mesenchymal-to-vasculogenic" phenotypic transition as an essential step in metastatic progression.

Na+/H+ exchanger regulatory factor 1 expression levels in blood and tissue predict breast tumour clinical behaviour.

AIMS: Several studies have demonstrated that Na(+) /H(+) exchanger regulatory factor 1 (NHERF1) protein, which is overexpressed and heterogeneously distributed in different stages of breast cancer, could be used as a tumour marker for prognosis in molecular detection strategies. We observed that tumour-infiltrated lymphocytes in the tumour tissue display a high level of NHERF1 staining, in contrast to those present in the contiguous non-involved tissue. Hypothesizing that cancer cells elicit a specific T-cell response associated with the characteristics of the solid tumour, our aim was to evaluate NHERF1 in peripheral lymphocytes from healthy donors and breast cancer patients. METHODS AND RESULTS: NHERF1 levels were analysed in 55 breast cancer patients and 40 healthy donors, and these levels were compared with clinical pathological features. NHERF1 was overexpressed in circulatory peripheral lymphocytes from patients as compared with those from healthy subjects. Furthermore, in both circulatory lymphocytes and tissues, NHERF1 was positively associated with tumour grade, Nottingham Prognostic Index and oestrogen receptor, whereas there was no association with other clinical parameters in either tissue. CONCLUSIONS: We propose that NHERF1 measurements in circulatory lymphocytes of breast cancer patients may be a valid method for the prediction of breast cancer occurrence and prognosis, and may have value in the management of cancer patients.

NHERF1/EBP50 in Breast Cancer: Clinical Perspectives.

Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression has been demonstrated to be altered in breast cancer, but its role in mammary cancerogenesis and progression remains still undefined. In this paper, we review what is known on the pathological role and the potential clinical application of NHERF1 protein in breast cancer. Recent evidence shows that an increased cytoplasmic expression of NHERF1 suggests a key role of its localization/compartmentalization in defining cancerogenesis, progression, and invasion. NHERF1 overexpression is associated with increasing tumor cytohistological grade, aggressive clinical behavior, unfavorable prognosis, and increased tumor hypoxia. Moreover, NHERF1 co-localizes with the oncogenic receptor HER2/neu in HER2/neu-overexpressing carcinoma and in distant metastases. These data make NHERF1 also a potential candidate of clinical relevance for anti-HER2/neu therapy.

NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space.

Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires membrane and released proteases focalized at membrane structures called invadopodia. While extracellular acidification is important in driving tumor invasion, the structure/function mechanisms underlying this regulation are still unknown. Invadopodia are similar in structure and function to osteoclast podosomes responsible for bone degradation, and extracellular acidification is central to podosome action, suggesting that it could also be for invadopodial function. Here, utilizing a novel system for in situ zymography in native matrices, we show that the Na(+)/H(+) exchanger (NHE1) and NHE1-generated extracellular acidification are localized at and necessary for invadopodial-dependent ECM degradation, thereby promoting tumor invasion. Stimulation with EGF increased both NHE1-dependent proton secretion and ECM degradation. Manipulation of the NHE1 expression by RNA interference or activity via either transport-deficient mutation or the specific inhibitor cariporide confirmed that NHE1 expression and activity are required for invadopodia-mediated ECM degradation. Taken together, our data show a concordance among NHE1 localization, the generation of a well-defined acidic extracellular pH in the nanospace surrounding invadopodia, and matrix-degrading activity at invadopodia of human malignant breast carcinoma cells, providing a structural basis for the role of NHE1 in invasion and identifying NHE1 as a strategic target for therapeutic intervention.

Biological role of NHERF1 protein expression in breast cancer.

AIMS: To determine the role of Na+/H+ exchanger regulatory factor (NHERF1) in breast cancerogenesis and progression. METHODS AND RESULTS: NHERF1 expression was examined in normal tissue, ductal carcinoma in situ (DCIS), invasive carcinoma (IBC), synchronous metastatic lymph node and metachronous distant metastases of a retrospective series of breast cancers. Fifty-one IBC, 42 DCIS and normal tissues were examined immunohistochemically, and the colocalization between NHERF1 and HER2/neu was studied by immunofluorescence. NHERF1 showed a different localization and pattern of expression in the different compartments of the breast. The mean value of cytoplasmic NHERF1 expression in paired samples was significantly higher in DCIS, IBC, distant metastases and metastatic lymph nodes with respect to normal tissues. Moreover, in metastatic lymph nodes NHERF1 was exclusively cytoplasmic. In the membrane NHERF1 was colocalized with overexpressed HER2/neu in DCIS, IBC and distant metastases. CONCLUSIONS: Breast cancerogenesis is characterized by increased cytoplasmic expression of NHERF1 as the tumour progresses, suggesting a role in this process. The switch from apical membranous to cytoplasmic expression is compatible with a dual role for NHERF1 as a tumour suppressor or tumour promoter dependent on its subcellular localization.

HPV16 E7-dependent transformation activates NHE1 through a PKA-RhoA-induced inhibition of p38alpha.

BACKGROUND: Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/(+)H(+) exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice. METHODOLOGY: Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1. PRINCIPAL FINDINGS: We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition. CONCLUSIONS: All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation.

RhoA protein expression in primary breast cancers and matched lymphocytes is associated with progression of the disease.

RhoA protein is over-expressed in breast cancer and other solid tumors and has been used in tumor biopsies as a quantitative tumor marker for progression, stage and prognosis in molecular detection strategies. Measuring protein markers in plasma or blood cells is preferred to tumor biopsies as it represents a minimally invasive, repeatable measurement that can be followed over time. In this study we evaluated the hypothesis that quantitative RhoA protein expression in circulatory lymphocytes is identically associated with the same tumor clinico-pathological features found in biopsies. RhoA protein levels were analyzed by Western blotting in circulating lymphocytes isolated from 52 consecutive patients with breast cancer and in 34 paired breast tumor biopsies from the same case study, and compared with the following clinico-pathological features of the patients: histological grade, tumor size, steroid receptor status, lymphonode status, proliferative activity and prognosis [Nottingham Prognostic Index (NPI)]. We observed that the level of circulatory, peripheral lymphocyte RhoA expression reflected that found in the matched biopsy of the same patient. Furthermore, similarly to previous reports regarding breast cancer tissue biopsies, the level of RhoA protein expression in both biopsies and in circulatory lymphocytes was positively associated with tumor size, grade, proliferative activity of the tumor biopsy and NPI, while there was no significant association of RhoA protein expression with either estrogen- or progesterone-receptor expression. Our study demonstrated that the association of lymphocyte RhoA protein expression with classical clinico-pathological parameters closely corresponded with that observed for RhoA protein expression in the tumor biopsies. We propose that measurement of RhoA expression in the circulatory lymphocytes of breast cancer patients can be used to predict breast cancer occurrence, progression and prognosis and may prove valuable in the management of cancer patients.

The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells.

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.

Cytoskeleton and paclitaxel sensitivity in breast cancer: the role of beta-tubulins.

The antineoplastic effect of paclitaxel is mainly related to its ability to bind the beta subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I beta-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of beta-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of beta-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC(50). The Class I beta-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III beta-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III beta-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between beta-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III beta-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III beta-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients.

The complexity of targeting EGFR signalling in cancer: from expression to turnover.

The epidermal growth factor receptor (ErbB1 or EGFR) has been found to be altered in a variety of human cancers. A number of agents targeting these receptors, including specific antibodies directed against the ligand-binding domain of the receptor and small molecules that inhibit kinase activity are either in clinical trials or are already approved for clinical treatment. However, identifying patients that are likely to respond to such treatments has been challenging. As a consequence, it still remains important to identify additional alterations of the tumor cell that contribute to the response to EGFR-targeted agents. While EGFR-mediated signalling pathways have been well established, there is still a rather limited understanding of how intracellular protein-protein interactions, ubiquitination, endocytosis and subsequent degradation of EGFR contribute to the determination of sensitivity to EGFR targeting agents and are emerging areas of investigation. This review primarily focuses on the basic signal transduction pathways mediated through activated membrane bound and/or endosomal EGFR and emphasizes the need to co-target additional proteins that function either upstream or downstream of EGFR to improve cancer therapy.

Protein kinase A gating of a pseudopodial-located RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell lines.

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.

The Na+-H+ exchanger-1 induces cytoskeletal changes involving reciprocal RhoA and Rac1 signaling, resulting in motility and invasion in MDA-MB-435 cells.

INTRODUCTION: An increasing body of evidence shows that the tumour microenvironment is essential in driving neoplastic progression. The low serum component of this microenvironment stimulates motility/invasion in human breast cancer cells via activation of the Na+-H+ exchanger (NHE) isoform 1, but the signal transduction systems that underlie this process are still poorly understood. We undertook the present study to elucidate the role and pattern of regulation by the Rho GTPases of this serum deprivation-dependent activation of both NHE1 and subsequent invasive characteristics, such as pseudopodia and invadiopodia protrusion, directed cell motility and penetration of normal tissues. METHODS: The present study was performed in a well characterized human mammary epithelial cell line representing late stage metastatic progression, MDA-MB-435. The activity of RhoA and Rac1 was modified using their dominant negative and constitutively active mutants and the activity of NHE1, cell motility/invasion, F-actin content and cell shape were measured. RESULTS: We show for the first time that serum deprivation induces NHE1-dependent morphological and cytoskeletal changes in metastatic cells via a reciprocal interaction of RhoA and Rac1, resulting in increased chemotaxis and invasion. Deprivation changed cell shape by reducing the amount of F-actin and inducing the formation of leading edge pseudopodia. Serum deprivation inhibited RhoA activity and stimulated Rac1 activity. Rac1 and RhoA were antagonistic regulators of both basal and stimulated tumour cell NHE1 activity. The regulation of NHE1 activity by RhoA and Rac1 in both conditions was mediated by an alteration in intracellular proton affinity of the exchanger. Interestingly, the role of each of these G-proteins was reversed during serum deprivation; basal NHE1 activity was regulated positively by RhoA and negatively by Rac1, whereas RhoA negatively and Rac1 positively directed the stimulation of NHE1 during serum deprivation. Importantly, the same pattern of RhoA and Rac1 regulation found for NHE1 activity was observed in both basal and serum deprivation dependent increases in motility, invasion and actin cytoskeletal organization. CONCLUSION: Our findings suggest that the reported antagonistic roles of RhoA and Rac1 in cell motility/invasion and cytoskeletal organization may be due, in part, to their concerted action on NHE1 activity as a convergence point.

Molecular and functional characteristics of erbB2 in normal and cancer breast cells.

The expression pattern of erbB2 and its transmembrane polymorphisms (Ile654Val and Ile655Val) were investigated in a panel of human normal and neoplastic breast cell lines to evaluate whether the expression pattern was affected by changes in the gene structure. At least two peptides of lower molecular mass forms (95 and 68 kDa) than the holoreceptor (185 kDa), comprehensive of the tyrosine kinase domain, were detected in all cells. Both peptides were also phosphorylated, suggesting a functional role in signal transduction. The presence of the polymorphisms found in two cell lines was unrelated to the expression of the lower molecular mass proteins.