The Adaptability of Chromosomal Instability in Cancer Therapy and Resistance.

Variation in chromosome structure is a central source of DNA damage and DNA damage response, together representinga major hallmark of chromosomal instability. Cancer cells under selective pressure of therapy use DNA damage and DNA damage response to produce newfunctional assets as an evolutionary mechanism. Recent efforts to understand DNA damage/chromosomal instability and elucidate its role in initiation or progression of cancer have also disclosed its vulnerabilities represented by inappropriate DNA damage response, chromatin changes, andinflammation. Understanding these vulnerabilities can provide important clues for predicting treatment response and for the development of novel strategies that prevent the emergence of therapy resistant tumors.

The pattern of gene copy number alteration (CNAs) in hepatocellular carcinoma: an in silico analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver cancer that occurs predominantly in patients with previous liver conditions. In the absence of an ideal screening modality, HCC is usually diagnosed at an advanced stage. Recent studies show that loss or gain of genomic materials can activate the oncogenes or inactivate the tumor suppressor genes to predispose cells toward carcinogenesis. Here, we evaluated both the copy number alteration (CNA) and RNA sequencing data of 361 HCC samples in order to locate the frequently altered chromosomal regions and identify the affected genes. RESULTS: Our data show that the chr1q and chr8p are two hotspot regions for genomic amplifications and deletions respectively. Among the amplified genes, YY1AP1 (chr1q22) possessed the largest correlation between CNA and gene expression. Moreover, it showed a positive correlation between CNA and tumor grade. Regarding deleted genes, CHMP7 (chr8p21.3) possessed the largest correlation between CNA and gene expression. Protein products of both genes interact with other cellular proteins to carry out various functional roles. These include ASH1L, ZNF496, YY1, ZMYM4, CHMP4A, CHMP5, CHMP2A and CHMP3, some of which are well-known cancer-related genes. CONCLUSIONS: Our in-silico analysis demonstrates the importance of copy number alterations in the pathology of HCC. These findings open a door for future studies that evaluate our results by performing additional experiments.

DNA Damage Response Protein CHK2 Regulates Metabolism in Liver Cancer.

Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. SIGNIFICANCE: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma.

Defective mitosis-linked DNA damage response and chromosomal instability in liver cancer.

Hepatocellular carcinoma (HCC), the most common form of liver cancer, represents a health problem in hepatic viruses-eradicating era because obesity, type 2 diabetes, and nonalcoholic steatohepatitis (NASH) are considered emerging pathogenic factors. Metabolic disorders underpin mitotic errors that lead to numerical and structural chromosome aberrations in a significant proportion of cell divisions. Here, we review that genomically unstable HCCs show evidence for a paradoxically DNA damage response (DDR) which leads to ongoing chromosome segregation errors. The understanding of DDR induced by defective mitoses is crucial to our ability to develop or improve liver cancer therapeutic strategies.

CHK2 overexpression and mislocalisation within mitotic structures enhances chromosomal instability and hepatocellular carcinoma progression.

OBJECTIVE: Chromosomal instability (CIN) is the most common form of genomic instability, which promotes hepatocellular carcinoma (HCC) progression by enhancing tumour heterogeneity, drug resistance and immunity escape. CIN per se is an important factor of DNA damage, sustaining structural chromosome abnormalities but the underlying mechanisms are unknown. DESIGN: DNA damage response protein checkpoint kinase 2 (Chk2) expression was evaluated in an animal model of diethylnitrosamine-induced HCC characterised by DNA damage and elevated mitotic errors. Chk2 was also determined in two discrete cohorts of human HCC specimens. To assess the functional role of Chk2, gain on and loss-of-function, mutagenesis, karyotyping and immunofluorescence/live imaging were performed by using HCT116, Huh7 and human hepatocytes immortalised with hTERT gene (HuS). RESULTS: We demonstrate that mitotic errors during HCC tumorigenesis cause lagging chromosomes/DNA damage and activation of Chk2. Overexpression/phosphorylation and mislocalisation within the mitotic spindle of Chk2 contributes to induce lagging chromosomes. Lagging chromosomes and mitotic activity are reversed by knockdown of Chk2. Furthermore, upregulated Chk2 maintains mitotic activity interacting with Aurora B kinase for chromosome condensation and cytokinesis. The forkhead-associated domain of Chk2 is required for Chk2 mislocalisation to mitotic structures. In addition, retinoblastoma protein phosphorylation contributes to defective mitoses. A cohort and independent validation cohort show a strong cytoplasm to nuclear Chk2 translocation in a subset of patients with HCC. CONCLUSIONS: The study reveals a new mechanistic insight in the coinvolvement of Chk2 in HCC progression. These findings propose Chk2 as a putative biomarker to detect CIN in HCC providing a valuable support for clinical/therapeutical management of patients.

New molecular targets for functionalized nanosized drug delivery systems in personalized therapy for hepatocellular carcinoma.

Hepatocellular carcinoma, the most frequent solid tumor of the liver, has a very poor prognosis, being the second most common cause of death from cancer worldwide. The incidence and mortality of this liver tumor are increasing in most areas of the world as a consequence of aging and the emerging of new risk factors such as the metabolic syndrome, beside the recognized role of hepatitis B and C viral infections and alcohol abuse. Despite the increasing knowledge on the molecular mechanisms underlying hepatic carcinogenesis, effective therapeutic strategies are still an unmet clinical need. Efforts have been made to develop selective drugs as well as effective targeted drug delivery systems. The development of novel drug carriers for therapeutic molecules can indeed offer a valuable strategy to ameliorate the efficacy of HCC treatment. In this review, we discuss recent drug delivery strategies for HCC treatment based on the exploitation of targeted nanoparticles (NPs). Indeed, a few of these platforms have achieved an advanced stage of preclinical development. Here, we review the most promising drug nanovehicles based on both synthetic and natural polymers, including polysaccharides that have emerged for their biocompatibility and biodegradability. To maximize site-selectivity and therapeutic efficacy, drug delivery systems should be functionalized with ligands which can specifically recognize and bind targets expressed by HCC, namely cell membrane associated antigens, receptors or biotransporters. Cell surface and intracellular molecular targets are exploited either to selectively deliver drug-loaded nanovehicles or to design novel selective therapeutics. In conclusion, the combination of novel and safe drug delivery strategies based on site-specific targeted drug nanovehicles with therapeutic molecular targets may significantly improve the pharmacological efficacy for the treatment of HCC.

Tumor Microenvironment, a Paradigm in Hepatocellular Carcinoma Progression and Therapy.

Hepatocellular carcinoma (HCC) is among the most lethal and prevalent cancers in the human population. Different etiological factors such as hepatitis B and C virus, alcohol and diabetes cause liver injury followed by inflammation, necrosis and hepatocytes proliferation. Continuous cycles of this destructive-regenerative process culminates in liver cirrhosis which is characterized by regenerating nodules that progress to dysplastic nodules and ultimately HCC. Despite its significance, there is only an elemental understanding of the pathogenetic mechanisms, and there are only limited therapeutic options. Therefore, the study of the involved molecular mechanisms can open a new insight to define more effective treatment strategies. A variety of alterations have been reported in HCC patients, particularly the cancer-associated microenvironment components including immune cells, fibroblast cells, endothelial cells and extracellular matrix can support the neoplastic cells to proliferate, growth and invade. This review summarizes the current state of knowledge and highlights the principal challenges that are relevant to controlling this milieu.

Determination and Characterization of Tetraspanin-Associated Phosphoinositide-4 Kinases in Primary and Neoplastic Liver Cells.

Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells.

CD63 tetraspanin is a negative driver of epithelial-to-mesenchymal transition in human melanoma cells.

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFß. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

Tetraspanin-enriched microdomains and hepatocellular carcinoma progression.

As in many tumors, heterogeneity within the cell population is one of the main features of hepatocellular carcinoma (HCC). Heterogeneity results from the ability of tumor to produce multiple subpopulations of cells with diverse genetic, biochemical and immunological characteristics. Little is known about how heterogeneity emerges and how it is maintained. Fluctuations in single cells can be masked or completely misrepresented when cell populations are analyzed. It has become exceedingly apparent that the utility of measurement based on the analysis of bulk specimens is limited by intra-tumor genetic and epigenetic heterogeneity, as characteristics of the most abundant cell type might not necessarily predict the properties of cell populations. Yet, such non-uniformities often unveil molecular patterns that can represent mechanisms of tumor progression. Interestingly, variability among single cells in a population may arise from different responses to intrinsic and extrinsic perturbations mainly mediated by the plasma membrane. The association of certain proteins, including tetraspanins, and lipids in specific location on the plasma membrane constitutes specialized structure called tetraspanin-enriched microdomains (TEMs). TEMs organization in cancer may reveal essential clues for understanding pathogenic mechanisms underlying cancer progression. Along these lines, TEMs and HCC progression represent a valuable paradigm for gaining a deeper understanding of such mechanisms.

Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third leading cause of cancer death. Recent epidemiological data indicate that the mortality rate of HCC will double over the next decades in the USA and Europe. Liver cancer progresses in a large percentage of cases during the clinical course of chronic fibro-inflammatory liver diseases leading to cirrhosis. Therefore, HCC development is regarded as the result of different environmental risk factors each involving different genetic, epigenetic- and chromosomal alterations and gene mutations. During tumour progression, the malignant hepatocytes and the activated hepatic stellate cells are accompanied by cancer-associated fibroblasts, myofibroblasts and immune cells generally called tumour stromal cells. This new and dynamic milieu further enhances the responsiveness of tumour cells towards soluble mediators secreted by tumour stromal cells, thus directly affecting the malignant hepatocytes. This results in altered molecular pathways with cell proliferation as the most important mechanism of liver cancer progression. Given this contextual complexity, it is of utmost importance to characterize the molecular pathogenesis of HCC, and to identify the dominant pathways/drivers and aberrant signalling pathways. This will allow an effective therapy for HCC that should combine strategies affecting both cancer and the tumour stromal cells. This review provides an overview of the recent challenges and issues regarding hepatic stellate cells, extracellular matrix dynamics, liver fibrosis/cirrhosis and therapy, tumour microenvironment and HCC.

The fibrotic microenvironment as a heterogeneity facet of hepatocellular carcinoma.

It has long been recognized that hepatocellular carcinoma heterogeneity arises from variation in the microenvironment or from genomic alteration. Only recently it has become clear that non-genetic alterations, such as cytoskeletal rearrangement, protein localization and formation of protein complexes, are also involved in generating phenotype variability. These proteome fluctuations cause genetically identical cells to vary significantly in their responsiveness to microenvironment stimuli. In the cirrhotic liver pre-malignant hepatocytes are continuously exposed to abnormal microenvironments, such as direct contact with activated hepatic stellate cells (HSCs) and extracellular matrix components. These abnormal environments can have pronounced influences on the epigenetic aspects of cells, translating into abnormal phenotypes. Here we discuss non-genetic causes of phenotypic heterogeneity of hepatocellular carcinoma, with an emphasis on variability of membrane protein complexes and transferred functions raising important implications for diagnosis and treatment.

Anticancer activity of an antisense oligonucleotide targeting TRADD combined with proteasome inhibitors in chemoresistant hepatocellular carcinoma cells.

Chemoresistance is a major cause of mortality of patients with advanced and metastatic hepatocellular carcinoma (HCC), the fifth most common cancer in the world. We employed a molecular approach to inhibit cell proliferation and induce apoptosis in HepG2 cells, originated from human hepatocarcinoma. TRADD gene expression was knocked down by an antisense oligonucleotide (ASO TRADD), resulting in TRADD protein decrease by 60%, coinciding with increase of apoptotic cell death of up to 30%. Combination of the ASO TRADD with the cytotoxic drugs 5-fluorouracil or paclitaxel did not improve chemosensitivity of HepG2 cells, while the combined administration of the ASO TRADD with proteasome inhibitors MG132 or ALLN inhibited cell proliferation by 80% and 93%, respectively. Taken together, these findings reveal the importance to combine proteasome inhibitors with silencing of anti-apoptotic signalling components to target HCC cells effectively and provide useful data for developing potential treatments of HCC.

Myristoylated Alanine-Rich protein Kinase C Substrate (MARCKS) expression modulates the metastatic phenotype in human and murine colon carcinoma in vitro and in vivo.

Several actin-binding proteins have been shown to be altered in metastatic cell lines and tumours and, in particular, Myristoylated Alanine-Rich protein Kinase C substrate (MARCKS) has been implicated in the pathogenesis of various highly metastatic epithelial malignancies. Considering that a large percentage of deaths due to colorectal cancer (CRC) are consequent to hepatic metastasization, aim of this study was to elucidate the involvement and mechanism of MARCKS in CRC by employing in vitro and in vivo approaches. Loss-of and-gain-on function approaches of MARCKS were employed in two human CRC cell lines: Clone A cells expressing MARCKS and LoVo cells known to have a frameshift mutation of MARCKS i.e. typically for MSI-H CRC. The data unveiled that altering MARCKS expression suppresses cell motility and invasion in human colon carcinoma cells when conditioned medium of liver-specific stromal cells (hepatic stellate cells) was used as chemoattractant. Depletion or re-expression of MARCKS inhibited proliferation with a reduction in expression of the mitotic regulator Aurora B kinase (AURKB), whereas AURKB-depletion did not modify MARCKS expression. In murine colon carcinoma CT26 cells, shRNA MARCKS-depletion reduced motility and invasion, and induced an aberrant, prolonged mitotic process. Significantly less metastases were produced in a syngeneic model of colon metastasis by MARCKS-depleted CT26 in comparison to CT26-tumour challenged mice. In conclusion, MARCKS plays an articulated role in the progression of colorectal cancer and might represent a suitable target to interfere and overcome the invasive behaviour of colon carcinoma cells at primary and distant sites.

The metastatic process: methodological advances and pharmacological challenges.

The metastatic spread of cancer is still the major barrier to the treatment of this disease. Understanding the molecular mechanisms underlying the metastatic process is of crucial importance to tune novel therapeutic strategies aimed at contrasting the dissemination of cancer. Metastasis is a sequential multistep process that ultimately leads to the cancer's outgrowth in a different organ from which it had originated. This clinically and experimentally involves the following steps: invasion of adjacent tissues, intravasation, transport of cancer cells through the circulatory system, arrest at a secondary site, extravasation and growth in a secondary organ. Additionally, tumor growth and metastasis depend on the ability of the tumor to induce its own blood supply through angiogenesis. Each of these steps can potentially be targeted by therapeutic agents, but the limited knowledge regarding the molecular events of metastasis makes most therapeutic strategies largely inefficient. However, important methodological advances have recently led to further insights into the biology of metastasis, thus raising the possibility of designing more appropriate pharmacological strategies to contrast the specific steps of the metastatic process. A variety of pharmacological approaches including inhibition of tumor invasion, angiogenesis, signal transduction pathways, and most recently the targeting of tumor stroma, are now under fervent development. Benefits and limits of these approaches, as well as, new therapeutic opportunities are herein discussed. Agents that limit any phase of the metastatic process may be therapeutically useful. Therefore, the future pharmacological challenge will be to combine drugs that target different aspects of this complex multistep process.

Tetraspanin CD81-regulated cell motility plays a critical role in intrahepatic metastasis of hepatocellular carcinoma.

BACKGROUND & AIMS: Human hepatocellular carcinoma (HCC) can invade the portal vein and metastasize to other parts of the liver. Currently, the molecular and cellular mechanisms underlying intrahepatic metastasis of HCC are poorly understood. Tumor invasiveness could be considered an aspect of dysregulated motility, and the mechanisms that inhibit cell movement are considered to counteract the spreading of cancer cells through the liver. Accumulating observations suggest that the CD81 tetraspanin may have an inhibitory effect on cell movement. METHODS: In the present study using both loss- and gain-of-gene function approaches, we verified that the functional interaction of tetraspanin CD81 with type II phosphoinositide 4-kinase (PI4KII) suppressed HCC cell motility by promoting the formation of CD81-enriched vesicles, non-endosomal intracellular structures, that sequestered actinin-4 with consequent remodeling of actin cytoskeleton. RESULTS: We reported that HCC cells expressing CD81 showed an inability to metastasize compared with HCC cells with undetectable levels of CD81. CONCLUSIONS: Taken together, these findings indicate that CD81 functions as a molecular organizer of membrane microdomains, whereby proteins such as PI4KII control actin remodeling and cell motility, establishing a role for these genes as negative modifiers of oncogenicity and HCC progression.

MARCKS is a downstream effector in platelet-derived growth factor-induced cell motility in activated human hepatic stellate cells.

Myristoylated alanine-rich protein kinase c substrate (MARCKS) has been suggested to be implicated in cell adhesion, secretion, motility and mitogenesis through regulation of the actin cytoskeletal structure. In the present study, a possible link between MARCKS and the platelet-derived growth factor (PDGF) signaling pathway was investigated in activated human hepatic stellate cells (hHSC), critical regulators of hepatic fibrogenesis. PDGF-BB stimulation resulted in a bi-directional movement of MARCKS that coincided with the phosphorylation of MARCKS and the activation of both PKCepsilon and PKCalpha. Biochemical inhibition of PKC kinase activity and small interfering RNA (siRNA) against PKCepsilon demonstrated that PKCepsilon is indispensable for PDGF-BB-induced MARCKS phosphorylation and cell migration. Immunoprecipitation studies revealed an association between MARCKS and the PDGFbeta-receptor, while the PDGFbeta-receptor and PKCalpha associated with focal adhesion kinase (FAK). Transient transfection with MARCKS DNA plasmid remarkably reduced PDGF-BB stimulated cell motility. In contrast, siRNA against MARCKS increased cell migration in RNAi treated cells in comparison to the scrambled control cells. In conclusion, the present study indicates that MARCKS play a major key role in PDGF-BB-induced chemotaxis in activated hHSC.

Farnesyltransferase inhibitor, ABT-100, is a potent liver cancer chemopreventive agent.

PURPOSE: Treatment of hepatocellular carcinoma raised on cirrhotic liver represents a major endeavor because surgery and chemotherapeutic management fail to improve the clinical course of the disease. Chemoprevention could represent an important means to inhibit the process of hepatocarcinogenesis. Farnesyltransferase inhibitors are a class of drugs blocking the growth of tumor cells with minimal toxicity towards normal cells. EXPERIMENTAL DESIGN: In the present study, we investigated the effects of a novel farnesyltransferase inhibitor, ABT-100, on human liver cancer cell lines, HepG2 and Huh7, and on an animal model of hepatocarcinogenesis. RESULTS: ABT-100 inhibited HepG2 and Huh7 cell growth as well as the invading ability of Huh7 on Matrigel. In HepG2 and Huh7 cells, ABT-100 inhibited growth factor-stimulated phosphoinositide 3-kinase and Akt/protein kinase B activity. Furthermore, ABT-100 inhibited Akt-dependent p27(Kip1) phosphorylation and this event was associated with increased levels of p27(Kip1) in the nucleus and reduced activity of the cyclin-dependent kinase 2. Moreover, ABT-100 treatment resulted in a significant reduction in tumor incidence and multiplicity. CONCLUSIONS: Taken together, these findings identify a mechanism of ABT-100 function and show the efficacy of ABT-100 as a chemopreventive agent of hepatocellular carcinoma.

Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells.

The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage.

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen-sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR).

We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying alpha6beta4 expression, suggesting an interference with the invasion process by androgens. Here, we investigated EGF-mediated signal transduction processes that lead to invasion in PC3-AR cells. We show that EGF-induced EGFR autotransphosphorylation is reduced in PC3-AR cells compared to PC3 cells transfected only with the vector (PC3-Neo). EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, and EGF-PI3K interaction are also decreased in PC3-AR cells and further reduced by treatment with androgen. Finally, we show that EGFR internalization process was reduced in PC3-AR and LNCaP cells compared to PC3-Neo. Investigations on the location of AR in PC3-AR transfected cells were also conducted. Immunoconfocal microscopy and coimminoprecipitation studies demonstrated the presence of an interaction between EGFR and AR at membrane level in PC3-AR and LNCaP cells. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less-malignant phenotype by interfering with EGFR signaling leading to invasion through a mechanism involving an interaction between AR and EGFR.