P-cadherin Overexpression Research Articles

Abstract 310: Cadherin-dependent modulation of chemosensitivity in oral tumor progression

Abstract The progression of healthy oral epithelia to an oral carcinoma often involves aberrant expression of a group of calcium-dependent transmembrane proteins called cadherins. Histological studies of human tumors have demonstrated increased expression of the epithelial-resident placental (P-cadherin) and ectopic expression of the mesenchymal neuronal (N-cadherin) cadherin in oral dysplasia. We hypothesized that these changes in cadherin expression during early oral tumor development could facilitate increases in survival signaling. We genetically modified dysplastic oral keratinocytes to overexpress P-cadherin or suppress endogenous expression of N-cadherin. P-cadherin overexpression and N-cadherin suppression independently elicited a 3-6-fold decrease in apoptosis levels in response to docetaxel treatment compared to similarly treated controls. We found that in untreated dysplastic cells, P- and N-cadherin expression had positive and negative correlations, respectively, with the activation of the survival-associated enzymes focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and AKT kinase. The total levels of nuclear factor kappa B (NFkb) were positively correlated with P-cadherin overexpression and reciprocally correlated with levels of N-cadherin. In docetaxel-treated dysplastic cells, we observed the reverse effect compared to untreated cells: the increased level of N-cadherin was positively correlated with FAK activity whereas FAK activity was suppressed by P-cadherin. P-cadherin expression in docetaxel-treated cells also increased activation of both MAPK and AKT, whereas alteration of N-cadherin levels had no effect. Because N-cadherin levels are known to increase in late-stage oral tumors, we extended these signaling studies to oral squamous carcinoma cells (OSCCs) expressing low or high levels of N-cadherin. In contrast to its suppression of survival pathways in docetaxel-treated dysplastic cells, in docetaxel-treated OSCCs, N-cadherin expression strongly enhanced phosphorylation of MAPK and AKT, and increased expression of NFkb. N-cadherin overexpression during this treatment also increased expression of the antiapoptotic proteins Mcl-1 and suppressed phosphorylation of the apoptotic regulator signal transducer and activator of transcription 3 (pSTAT3). Increase cadherin expression in all untreated cell lines increased expression of an inducer of epithelial to mesenchymal transition, the transmembrane protein mucin-1 (MUC1). N-cadherin overexpression also increased motility in both cell lines. Taken together, our data demonstrate antiapoptotic signaling directed by increased levels of P-cadherin in oral dysplasia and N-cadherin in OSCC, whereas increased levels of N-cadherin in dysplastic cells directed cellular signaling that were associated with a proapoptotic phenotype. Citation Format: Aaron Mody, Cassandra Decker, Kathryn R. Lawson. Cadherin-dependent modulation of chemosensitivity in oral tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 310.

Breast cancer prognosis and P-cadherin expression: systematic review and study-level meta-analysis

ObjectiveP-cadherin can act both as a tumour suppressor and an oncogene. The clinical prognostic value of P-cadherin overexpression in breast cancer (BC) remains unclear. We conducted a study-level meta-analysis to...

Selected Articles from This Issue

Resistance to current lines of therap. in gastrointestinal stromal tumors (GIST) can occur through a variety of secondary mutations that occur in KIT. Novel therapeutics are still needed to address the mutational heterogeneity of KIT in GIST patients. In this study, Gupta and collaborators demonstrate the broad spectrum of ripretinib against primary and secondary drug-resistant mutations. Ripretinib is a “switch-control” kinase inhibitor designed to address even aggressively activated mutants to generate inactive conformations. Knowing that imatinib and MEK inhibition previously showed promise against imatinib-sensitive tumors, the authors also demonstrate the potency of combining ripretinib with MEKi in vitro and in vivo.KRAS mutations are present in up to 95% of pancreatic ductal adenocarcinoma (PDAC) cases. Inhibiting MEK (downstream of KRAS) would therefore appear to be a viable strategy for treating PDAC. Unfortunately, MEK inhibition as a monotherapy has been suboptimal in preclinical and clinical studies. Since CDKN2A mutations are also prevalent in a majority of PDAC tumors, Willobee and collaborators investigated the combination of CDK4/6 inhibitors with MEK inhibition. Combined CDK4/6 and MEK inhibition resulted in a significant survival benefit in an aggressive genetic mouse model of PDAC and provided a mechanistic rationale to explore the combination in clinical studies.For many cancers, the overexpression of P-cadherin is significantly correlated with lower overall and progression-free survival. Due to its limited expression in normal tissue and broad expression in tumors, Sheng and colleagues developed the P-cadherin specific antibody-drug conjugate PCA062. PCA062 contains a P-cadherin specific antibody bound to DM1 via a non-cleavable thioester linkage SMCC. The in vivo efficacy of PCA062 was demonstrated in cell- and patient-derived xenograft models of esophageal, triple-negative breast, and head and neck cancers. The authors note that limited efficacy signals were observed at dose escalation in Phase 1 trials. Taken together, the authors narrate the promise and challenges in targeting P-cadherin with the ADC modality.Inhibition of the programmed cell death ligand 1 (PD-1) axis has succeeded in many different solid tumors. The response of glioblastoma to PD-1 inhibition, however, remains limited due to its cold immune environment. Zhang and colleagues analyzed data from TCGA and clinical glioma samples and determined that Wnt/β-catenin expression was inversely related to the degree of immune cell infiltration and PD-L1 expression. They demonstrate that Wnt inhibition enhanced anti-PD-1 antibodies in a homograft mouse model by increasing lymphocyte infiltration and IFN-γ secretion. Their results demonstrate the potential for modifying the glioblastoma tumor microenvironment using Wnt inhibitors to potentiate immune checkpoint inhibition.

Increased expression of P-cadherin is an indicator of poor prognosis in breast cancer: a systematic review and meta-analysis.

P-cadherin (CDH3), located at 16q22.1 belonging to classical cadherin family, is a calcium-dependent glycoprotein associated with cell to cell adhesion, migration, and invasion in cancer. This meta-analysis was conducted to examine the prognostic utility of P-cadherin expression in breast cancer (BC). A comprehensive literature search was carried out using the available databases to obtain relevant research articles to test the relationship between P-cadherin and BC. Correlation of P-cadherin expression and disease-free survival (DFS) or overall survival (OS) was tested using hazard ratio (HR), relative risk (RR) at 95% confidence interval (CI) by univariate and/or multivariate analysis. A total of 11 studies from 7 countries were found to be relevant and were further subjected to statistical analysis to find an association between the P-cadherin expression with BC. Additionally, we have also performed a co-relation analysis of P-cadherin expression with GOBO and Cancertool in breast cancer using publicly available breast cancer datasets. Our study shows that P-cadherin expression is significantly linked with poor prognosis in the various subtypes of BC. The HR for OS and DFS was 1.87 (95% CI = 1.48-2.36) and 1.64 (95% CI = 1.18-2.27) respectively. In this meta-analysis, we identified a positive correlation between the overexpression of P-cadherin and BC. Our study demonstrates that P-cadherin overexpression can be used as a prognostic indicator in BC.

Prognositic significance of P-cadherin expression in breast cancer: Protocol for a meta-analysis.

Background:P-cadherin is a calcium-dependent cell–cell adhesion glycoprotein. It has been implicated in invasiveness and metastasis. However, the clinical prognostic value of overexpression of P-cadherin in patients with breast cancer (BC) remains unsettled.Methods:A systematic literature search will be performed in all available databases to quantitatively review eligible studies and identify all relevant data, which could be used to detect the relationship between overexpression of P-cadherin and overall survival (OS), disease-free survival (DFS), and clinicopathological parameters. Hazard ratio and 95% confidence intervals (CIs) or P value will be employed as effect measures to estimate the correlation between P-cadherin and the oncologic outcomes including overall survival (OS), disease-free survival (DFS). Odds ratios (ORs) and the 95% CIs will be evaluated for the pooled analysis of the correlation between P-cadherin expression and clinicopathological features. We will use the Review Manager (Revman) 5.3.5 software (Cochrane Community, London, United Kingdom) and STATA 14 software (version 14.0; Stata Corp, College Station, TX) to perform the meta-analysis to calculate the data.Results:The review will provide a high-quality synthesis of current evidence of the prognostic role of P-cadherin in BCs. The results will be published in a peer-reviewed journal.Conclusion:We hope that the results of this study will provide significant evidence to assess whether the expression of P-cadherin is associated with poor prognosis in patients with BC.PROSPERO registration number:This meta-analysis protocol has been registered in the PROSPERO network with registration number: CRD42019119880.

MOLECULAR AND GENETIC SUBTYPES OF OVARIAN CANCER: PROSPECTS FOR FURTHER STUDIES

High-grade ovarian carcinoma is a histopathological diagnosis, however, at the molecular level, ovarian cancer represents a heterogeneous group of diseases. Studies aimed at identifying molecular genetic subtypes of ovarian cancer are conducted in order to find the answer to the question: can different molecular subgroups influence the choice of treatment? One of the achievements in this trend is the recognition of the dualistic model that categorizes various types of ovarian cancer into two groups designated high-grade (HG) and low-grade (LG) tumors. However, the tumor genome sequencing data suggest the existence of 6 ovarian carcinoma subtypes, including two LG and four HG subtypes. Subtype C1 exhibits a high stromal response and the lowest survival. Subtypes C2 and C4 demonstrate higher number of intratumoral CD3 + cells, lower stromal gene expression and better survival than sybtype C1. Subtype C5 (mesenchymal) is characterized by mesenchymal cells, over-expression of N-cadherin and P-cadherin, low expression of differentiation markers, and lower survival rates than C2 and C4. The use of a consensus algorithm to determine the subtype allows identification of only a minority of ovarian carcinomas (approximately 25%) therefore, the practical importance of this classification requires additional research. There is evidence that it makes sense to randomize tumors into groups with altered expression of angiogenic genes and groups with overexpression of the immune response genes, as in the angiogenic group there is a comparative superiority in terms of survival. The administration of bevacizumab in the angiogenic group improves survival, while the administration of bevacizumab in the immune group even worsens the outcome. Molecular subtypes with worse survival rates (proliferative and mesenchymal) also benefit most from bevacizumab treatment. This review focuses on some of the advances in understanding molecular, cellular, and genetic changes in ovarian carcinomas with the results achieved so far regarding the formulation of molecular subtypes of ovarian cancer, however further studies are needed.

SRC inhibition prevents P-cadherin mediated signaling and function in basal-like breast cancer cells

BackgroundBasal-like breast cancer (BLBC) is a poor prognosis subgroup of triple-negative carcinomas that still lack specific target therapies and accurate biomarkers for treatment selection. P-cadherin is frequently overexpressed in these tumors, promoting cell invasion, stem cell activity and tumorigenesis by the activation of Src-Family kinase (SRC) signaling. Therefore, our aim was to evaluate if the treatment of BLBC cells with dasatinib, the FDA approved SRC inhibitor, would impact on P-cadherin induced tumor aggressive behavior.MethodsP-cadherin and SRC expression was evaluated in a series of invasive Breast Cancer and contingency tables and chi-square tests were performed. Cell-cell adhesion measurements were performed by Atomic Force Microscopy, where frequency histograms and Gaussian curves were applied. 2D and 3D cell migration and invasion, proteases secretion and self-renew potential were evaluated in vitro. Student’s t-tests were used to determine statistically significant differences. The cadherin/catenin complex interactions were evaluated by in situ proximity-ligation assay, and statistically significant results were determined by using Mann-Whitney test with a Bonferroni correction. In vivo xenograft mouse models were used to evaluate the impact of dasatinib on tumor growth and survival. ANOVA test was used to evaluate the differences in tumor size, considering a confidence interval of 95%. Survival curves were estimated by the Kaplan-Meier’s method, using the log-rank test to assess significant differences for mice overall survival.ResultsOur data demonstrated that P-cadherin overexpression is significantly associated with SRC activation in breast cancer cells, which was also validated in a large series of primary tumor samples. SRC activity suppression with dasatinib significantly prevented the in vitro functional effects of P-cadherin overexpressing cells, as well as their in vivo tumorigenic and metastatic ability, by increasing mice overall survival. Mechanistically, SRC inhibition affects P-cadherin downstream signaling, rescues the E-cadherin/p120-catenin complex to the cell membrane, recovering cell-cell adhesion function.ConclusionsIn conclusion our findings show that targeting P-cadherin/SRC signaling and functional activity may open novel therapeutic opportunities for highly aggressive and poor prognostic basal-like breast cancer.

Abstract CT128: Phase 1 study of FF-21101(90Y), a radioimmunotherapeutic (RIT) targeting P-cadherin, in advanced solid tumors

Abstract Background: Overexpression of P-cadherin, a calcium-dependent cell-to-cell adhesion glycoprotein, correlates with increased tumor cell invasiveness in breast, colon, lung and pancreatic tumors. FF-21101 is a human-mouse chimeric monoclonal antibody directed against P-cadherin, conjugated with 111In for dosimetry and biodistribution and 90Y for therapy. We report the biodistribution and preliminary anti-cancer activity of FF-21101(90Y) in a first-in-human study. Methods: Patients (pts) received 5 mCi/5mg FF-21101(111In) 1 week before the FF-21101(90Y) therapeutic dose to assess biodistribution and ensure 90Y radiation dose estimates did not exceed the maximum tolerated dose (MTD) for each organ. Single therapeutic dose cohorts were planned for FF-21101(90Y) (8 mCi/mg) at 5, 10, 15, 20 or 25 mCi/m2 (3+3 dose escalation schema), with repeat doses allowed every 4 months in pts demonstrating clinical benefit. Disease assessments were based on RECIST V1.1. Pre-treatment tumor samples were assessed for P-cadherin expression by immunohistochemistry (IHC). Pharmacokinetics (PK) of FF-21101 were also assessed. Results: Fourteen pts (6M, 8F) with advanced primary solid tumors and loco-regional metastases were treated in the 5 dose cohorts. Median (range) values: age 58 years (31 – 69), number of prior systemic treatments 3 (0 – 9), tumor types: soft-tissue sarcoma (3), ovarian carcinoma (CA) (2), and vaginal CA, pancreatic neuroendocrine tumor (PNET), desmoplastic small round cell tumor, colorectal CA, appendiceal CA, cholangiocarcinoma, squamous cell CA (SCC) of the anus, papillary thyroid CA and carcinoid (1 each). Primary and/or metastatic tumors from 12 of 14 pts (85.7%) demonstrated uptake of FF-21101(111In) with highest uptake seen in epithelial tumors, consistent with P-cadherin targeting. Median (range) time on study following the therapeutic dose was 21 (2 - 62) weeks, with 3 pts on study > 1 year (2 ovarian CA, 1 carcinoid). Four pts have received multiple treatments. One ovarian CA pt achieved a partial response (65% reduction); this pt also had the highest H score by IHC staining. High H scores (≥ 100) were demonstrated in 5 of 13 pts: 2 ovarian, 1 PNET, 1 SCC of the anus, 1 cholangiocarcinoma; 4 remained on study for 49 - 62 weeks, thus demonstrating the potential predictive utility of pre-treatment tumor P-cadherin expression. FF-21101(90Y) has been well-tolerated; the MTD has not been reached. The most frequently reported drug-related AEs include lymphopenia (n = 8), thrombocytopenia (n = 4) and leukopenia (n = 3); Gr 3 lymphopenia was observed in 3 pts, all reversible. There have been no drug-related serious AEs. Mean FF-21101 Cmax and AUC0-t increased with dose, suggesting linear PK. Conclusions: Tumor P-cadherin overexpression provides an attractive target for RIT. Radiolabeled FF-21101 exhibits favorable dosimetry and biodistribution, good tolerability and preliminary evidence of anti-cancer activity. Pre-treatment tumor P-cadherin expression may be an important biomarker for patient selection. Citation Format: Vivek Subbiah, William Erwin, Osama Mawlawi, Carlos Gonzalez-Lepera, Masahiko Tokura, Michael Kurman, Holly Liu, David S. Hong, Funda Meric-Bernstam, Shubham Pant, Mary Johansen, Timothy Madden, Elmer Santos, Gregory Ravizzini. Phase 1 study of FF-21101(90Y), a radioimmunotherapeutic (RIT) targeting P-cadherin, in advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT128.

Abstract CT097: Phase 1 study of FF-21101(90Y), a radioimmunotherapeutic targeting P-cadherin, in advanced solid tumors

Abstract Background: CDH3 gene overexpression of P-cadherin correlates with increased tumor cell invasiveness and is observed in breast, colon, lung, and pancreatic tumors. FF-21101 is a human-mouse chimeric monoclonal antibody directed against P-cadherin, conjugated with 111In for dosimetry and 90Y for therapy. This first-in-human study assesses dosimetry (biodistribution) and therapeutic outcome of FF-21101(90Y). Methods: For dosimetry, patients (pts) received 5 mCi/5mg FF-21101(111In) 1 week before the FF-21101(90Y) therapeutic dose to assess biodistribution and ensure 90Y radiation dose estimates did not exceed the recommended allowable limit for each organ. Single therapeutic dose cohorts were planned for FF-21101(90Y) (8 mCi/mg) at 5, 10, 15, 20 or 25 mCi/m2 (3+3 dose escalation schema), with repeat doses allowed every 4 cycles. Disease assessments were based on RECIST V1.1. Pre-treatment tumor samples were assessed for P-cadherin expression by immunohistochemistry (IHC). Pharmacokinetics (PK) of FF-21101 also were assessed. Results: Seven pts (3M, 4F) with advanced primary solid tumors and loco-regional metastases were treated with FF-21101(90Y) in the first 3 dose cohorts. Median (range) values: age 55 years (31 – 69), number of prior treatments, including surgery, radiation and/or chemotherapy 5 (2 – 8); tumor types: ovarian carcinoma (CA) (2 pts), vaginal CA, pancreatic neuroendocrine tumor (PNET), desmoplastic small round cell (DSRC) tumor, colorectal CA (CRC), recurrent liposarcoma (1 pt each). Primary and/or metastatic tumors from 4 of 7 pts (57.1%) demonstrated positive uptake of FF-21101(111In). Highest uptake was seen in epithelial tumors, consistent with P-cadherin targeting. Median (range) time on study following the FF-21101(90Y) dose was 8 (4 – 40) weeks. The vaginal CA pt demonstrated an 18.5% decrease as best response through 16 weeks following a single therapeutic dose. A viable pre-study tumor sample was not available for IHC staining. Pre-treatment tumor samples available from 6 of 7 patients demonstrated high H scores (≥ 100) in 3 pts (50%); 2 ovarian and 1 PNET; all remain on study through 4, 12 and 40 weeks, respectively, thus demonstrating the potential predictive utility of pre-treatment tumor P-cadherin expression. FF-21101(90Y) has been well-tolerated. Drug-related adverse events (AEs) include Gr 1 rash (1 pt) and increased AST (Gr 1/2, 2 pts) at 5 mCi/m2, and lymphopenia (1 Gr 3 in 3 pts at 10 mCi/m2), all reversible. There have been no drug-related serious AEs. Mean FF-21101 Cmax and AUC0-t increased with dose, suggesting linear PK. Conclusions: Tumor P-cadherin overexpression provides an attractive target for radioimmunotherapy. FF-21101(111In/90Y) exhibits favorable dosimetry, good tolerability and preliminary evidence of reduction in tumor burden. Pre-treatment tumor P-cadherin expression may be an important biomarker for patient selection. Citation Format: Vivek Subbiah, William Erwin, Osama Mawlawi, Carlos Gonzalez Lepera, Masahiko Tokura, Masayuki Kawakami, Holly Liu, Shubham Pant, Michele Rosner, Mary Johansen, Louis De Palatis, Thomas Myers, Linda Paradiso, Elmer Santos, Gregory Ravizzini. Phase 1 study of FF-21101(90Y), a radioimmunotherapeutic targeting P-cadherin, in advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT097. doi:10.1158/1538-7445.AM2017-CT097

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3β/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche.

Atomic force microscopy and graph analysis to study the P-cadherin/SFK mechanotransduction signalling in breast cancer cells.

Physical forces mediated by cell-cell adhesion molecules, as cadherins, play a crucial role in preserving normal tissue architecture. Accordingly, altered cadherins' expression has been documented as a common event during cancer progression. However, in most studies, no data exist linking pro-tumorigenic signaling and variations in the mechanical balance mediated by adhesive forces. In breast cancer, P-cadherin overexpression increases in vivo tumorigenic ability, as well as in vitro cell invasion, by activating Src family kinase (SFK) signalling. However, it is not known how P-cadherin and SFK activation impact cell-cell biomechanical properties. In the present work, using atomic force microscopy (AFM) images, cell stiffness and cell-cell adhesion measurements, and undirected graph analysis based on microscopic images, we have demonstrated that P-cadherin overexpression promotes significant alterations in cell's morphology, by decreasing cellular height and increasing its area. It also affects biomechanical properties, by decreasing cell-cell adhesion and cell stiffness. Furthermore, cellular network analysis showed alterations in intercellular organization, which is associated with cell-cell adhesion dysfunction, destabilization of an E-cadherin/p120ctn membrane complex and increased cell invasion. Remarkably, inhibition of SFK signaling, using dasatinib, reverted the pathogenic P-cadherin induced effects by increasing cell's height, cell-cell adhesion and cell stiffness, and generating more compact epithelial aggregates, as quantified by intercellular network analysis. In conclusion, P-cadherin/SFK signalling induces topological, morphological and biomechanical cell-cell alterations, which are associated with more invasive breast cancer cells. These effects could be further reverted by dasatinib treatment, demonstrating the applicability of AFM and cell network diagrams for measuring the epithelial biomechanical properties and structural organization.

Significance of P‐cadherin overexpression and possible mechanism of its regulation in intrahepatic cholangiocarcinoma and pancreatic cancer

It has become evident that P-cadherin, one of the classical cadherins, contributes to the malignant behavior of several types of cancer. In this study, we analyzed the expression of P-cadherin and its clinicopathological and prognostic values in intrahepatic cholangiocarcinoma (ICC) and pancreatic cancer. Furthermore, we investigated the functional role of P-cadherin in these cancer cells by knockdown and overexpression in vitro and by analyzing the correlation between the P-cadherin expression and its promoter methylation status. Thirty of 59 ICC cases (51%) and 36 of 73 pancreatic cancer cases (49%) stained positive for P-cadherin with mainly membranous distribution in tumor cells by immunohistochemistry. P-cadherin expression was significantly correlated with several clinicopathological factors, which reflect tumor behavior, and was identified as an independent adverse prognostic factor for disease-free survival in patients with ICC (relative risk [RR] 2.93, P = 0.04) and pancreatic cancer (RR 2.68, P = 0.005) via multivariate analyses. P-cadherin downregulation by siRNA suppressed migration and invasion, and P-cadherin overexpression induced the opposite effects in both ICC and pancreatic cancer cells, without any effects on cell proliferation. P-cadherin expression was related to its promoter methylation status in both cell lines and cancer tissues. In summary, P-cadherin overexpression may serve as a useful biomarker of invasive phenotype and poor prognosis; P-cadherin expression was found to be regulated by its promoter methylation. These results suggest that P-cadherin represents a novel therapeutic target for the treatment of ICC and pancreatic cancer.

P-cadherin potentiates ligand-dependent EGFR and IGF-1R signaling in dysplastic and malignant oral keratinocytes.

Oral and oropharyngeal cancer together constitute the sixth most common cancer worldwide, with over 400,000 new cases diagnosed each year. Early detection is paramount, as the 5-year survival rate for these cancers decreases markedly once tumors have become regionally invasive. In many tissues, including oral epithelia, neoplastic progression is accompanied by alterations in expression of the epithelial cell adhesion molecules E-cadherin and P-cadherin. Oral epithelia is one of only a few tissues in which P-cadherin levels have been noted to increase in dysplasia and well-differentiated carcinomas and decrease in advanced malignancies. In the present study, P-cadherin was overexpressed in both dysplastic and malignant oral keratinocytes to characterize the mechanisms by which aberrantly expressed P-cadherin may modulate tumor progression. We found that P-cadherin was able to potentiate ligand-dependent signaling of insulin-like growth factor 1 receptor (IGF-1R) in malignant keratinocytes and epidermal growth factor receptor (EGFR) in dysplastic cells. P-cadherin prolonged activation of the mitogen-activated protein kinase (MAPK) in both cell lines and also increased the magnitude of AKT phosphorylation in dysplastic cells. P-cadherin overexpression alone was sufficient to increase steady-state levels of the mesenchymal transcription factor Snail, increase cell motility and also induce morphological changes in dysplastic keratinocytes. Taken together, these data suggest that the aberrantly elevated levels of P-cadherin which occur in early oral tumor development may play a critical role in the augmentation of neoplastic signaling networks and in the further acquisition of aggressive phenotypes.

The basal epithelial marker P-cadherin associates with breast cancer cell populations harboring a glycolytic and acid-resistant phenotype.

BackgroundCancer stem cells are hypoxia-resistant and present a preponderant glycolytic metabolism. These characteristics are also found in basal-like breast carcinomas (BLBC), which show increased expression of cancer stem cell markers.Recently, we demonstrated that P-cadherin, a biomarker of BLBC and a poor prognostic factor in this disease, mediates stem-like properties and resistance to radiation therapy. Thus, the aim of the present study was to evaluate if P-cadherin expression was associated to breast cancer cell populations with an adapted phenotype to hypoxia.MethodsImmunohistochemistry was performed to address the expression of P-cadherin, hypoxic, glycolytic and acid-resistance biomarkers in primary human breast carcinomas. In vitro studies were performed using basal-like breast cancer cell lines. qRT-PCR, FACS analysis, western blotting and confocal microscopy were used to assess the expression of P-cadherin after HIF-1α stabilization, achieved by CoCl2 treatment. siRNA-mediated knockdown was used to silence the expression of several targets and qRT-PCR was employed to evaluate the effects of P-cadherin on HIF-1α signaling. P-cadherin high and low breast cancer cell populations were sorted by FACS and levels of GLUT1 and CAIX were assessed by FACS and western blotting. Mammosphere forming efficiency was used to determine the stem cell activity after specific siRNA-mediated knockdown, further confirmed by western blotting.ResultsWe demonstrated that P-cadherin overexpression was significantly associated with the expression of HIF-1α, GLUT1, CAIX, MCT1 and CD147 in human breast carcinomas. In vitro, we showed that HIF-1α stabilization was accompanied by increased membrane expression of P-cadherin and that P-cadherin silencing led to a decrease of the mRNA levels of GLUT1 and CAIX. We also found that the cell fractions harboring high levels of P-cadherin were the same exhibiting more GLUT1 and CAIX expression. Finally, we showed that P-cadherin silencing significantly decreases the mammosphere forming efficiency in the same range as the silencing of HIF-1α, CAIX or GLUT1, validating that all these markers are being expressed by the same breast cancer stem cell population.ConclusionsOur results establish a link between aberrant P-cadherin expression and hypoxic, glycolytic and acid-resistant breast cancer cells, suggesting a possible role for this marker in cancer cell metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-734) contains supplementary material, which is available to authorized users.

Abstract 4409: P-cadherin modulates signaling of multiple growth factor receptors and cellular aggressiveness in oral carcinoma cells

Abstract Aberrant expression of the epithelial cell adhesion protein P-cadherin has been observed to be independently causative and preventative with respect to tumor progression, depending on tissue type. Oral epithelial tumors have been shown to exhibit a unique signature of P-cadherin expression in which P-cadherin expression increases in dysplasia and is subsequently lost during malignancy. These studies suggest that early increases in P-cadherin expression may be critical events in tumor progression. Since E- and N-cadherin have been shown to physically interact with and modulate function of Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF) respectively, we hypothesized that P-cadherin could promote tumor development via its own interaction with receptor tyrosine kinases in a ligand-dependent manner. To identify functional interactions between P-cadherin and receptor tyrosine kinases, we retrovirally transduced two independently isolated oral squamous carcinoma cell lines to overexpress P-cadherin. Transduced cells were treated independently with one of seven different growth factors to identify alterations in mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways. For all experiments, protein was collected over the course of six hours at multiple timepoints to identify fluctuations in signaling. Although P-cadherin did not alter AKT phosphorylation, it was found to alter MAPK signaling in both a positive and negative manner. The most robust alterations in MAPK signaling occurred in response to activation of Insulin-like Growth Factor (IGF), Epithelial Growth Factor (EGF), and Keratinocyte Growth Factor (KGF). P-cadherin overexpression also induced phenotypic changes. Behavioral assays revealed that P-cadherin expression increased motility, but decreased expression of invasive proteases MMP-9 and MMP-14. P-cadherin cells grown in serum-free media for 48 hours exhibited increased cell number compared to control cells. A cell proliferation analysis (MTT assay) indicated lower rates of proliferation in P-cadherin -overexpressing cells compared to controls. This data suggests that the increased cell number of serum-starved P-cadherin cells was due to increased survival, rather than proliferation. These results demonstrate that P-cadherin overexpression can modulate ligand-dependent growth factor signaling and alter those cellular characteristics most commonly associated with tumor aggressiveness. Since P-cadherin expression has been shown to increase early on in the development of tumors, our research may provide a possible mechanism as to how this increase may play a role in tumor progression during dysplasia. Citation Format: James Mar, Adam Robinson, Rachel Neville, Desseree Lysne, Kathryn Lawson. P-cadherin modulates signaling of multiple growth factor receptors and cellular aggressiveness in oral carcinoma cells. [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 4409. doi:10.1158/1538-7445.AM2014-4409

Abstract 376: P-cadherin overexpression is associated with poor prognosis in pancreatic carcinoma and is regulated by its promoter hypomethylation

Abstract [Background] P-cadherin belongs to the family of classic cadherin, and accumulating evidences shows hat P-cadherin is strongly associated with carcinogenesis and confers alignant phenotype of various cancers. Pancreatic cancer has a poor prognosis, with an overall 5-year survival rate of only 5%. Despite advances in therapy recently, its survival rate still remains poor. This study aimed to analyze P-cadherin expression and its prognostic value in pancreatic carcinoma and to validate the P-cadherin gene (CDH3) promoter methylation status as the mechanism of this gene regulation. [Methods] Seventy-three patients diagnosed with pancreatic cancer were enrolled in this study. P-cadherin expression was examined by immunohistochemistry, and correlated with various clinicopathological features and prognosis. Furthermore, P-cadherin expression and its promoter methylation status were analyzed in pancreatic cancer cell lines. [Results] In pancreatic tissues, 50.7% (37/73) of cases shows a high expression of P-cadherin by immunohistochemical analysis, and high expression of P-cadherin was significantly correlated with gender, posterior tissue invasion and other organs invasion. In multivariate analyses, P-cadherin expression was the independent prognostic factors for disease-free survival (relative risk:2.68, p=0.005). P-cadherin expression was found to be related to its promoter methylation status in pancreatic cancer cell lines. Furthermore, when P-cadherin was down-regulated by specific siRNA, cell migration and invasion were significantly reduced. [Conclusion] These results suggested that expression of P-cadherin is a useful prognostic marker and seems to be important in the malignant behavior in pancreatic cancer. We are now conducting experiments to validate the CDH3 methylation status in the cancerous tissues and their adjacent normal counterpart, and investigating the biological role of P-cadherin in pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Keita Sakamoto, Katsunori Imai, Shigeki Nakagawa, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Takatoshi Ishiko, Toru Beppu, Hideo BaBa. P-cadherin overexpression is associated with poor prognosis in pancreatic carcinoma and is regulated by its promoter hypomethylation. [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 376. doi:10.1158/1538-7445.AM2014-376

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells.We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, β4 and β1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin in P-cadherin overexpression breast cancer models.

The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.

Abstract 3989: P-Cadherin overexpression modulates insulin-like growth factor signaling in oral squamous carcinoma cells.

Abstract Altered expression of cadherin cell adhesion proteins is a frequent occurrence in epithelial tumor development. P-cadherin, a component of normal epithelia, has been ascribed both causative and preventive roles in tumor development, contingent upon the tissue of origin. In oral epithelia, histological studies demonstrate an upregulation of P-cadherin during dysplasia, and a subsequent loss of P-cadherin in later stage oral squamous carcinomas. A possible mechanism by which P-cadherin may modulate tumor development is via alterations in ligand-mediated signaling of receptor tyrosine kinases, a phenomenon that has previously been demonstrated for both E- and N-cadherin. To better understand the role that P-cadherin plays in tumor development, retroviral transduction was used to deplete or overexpress P-cadherin in two oral squamous carcinoma cell lines. Six growth factors were independently utilized to screen for P-cadherin-dependent alterations in signaling, with the greatest effects occurring in response to insulin-like growth factor (IGF) and fibroblast growth factor (FGF). The increased level of P-cadherin in IGF-treated cells greatly altered the kinetics of mitogen-activated protein kinase (MAPK) signaling, but had little direct effect on the activation of the phosphoinositide 3-kinase (PI3K) pathway. P-cadherin overexpression increased steady-state levels of Snai1 protein, with further increases noted upon IGF administration. Inhibition of either MAPK or PI3K signaling pathways in P-cadherin overexpressing cells resulted in decreased Snai1 expression. Ser-9 phosphorylation of GSK-3β was unchanged after IGF treatment, suggesting P-cadherin contributes to Snai1 stability independent of GSK3β activity. P-cadherin over-expressing cells treated with IGF showed a time-dependent decrease in P-cadherin levels. This decrease was accompanied by a decrease in detectable levels of immunofluorescence staining of P-cadherin at cell borders. The internalization of P-cadherin could be inhibited by treatment with dynasore, a dynamin inhibitor. Also, increases in motility were found in P-cadherin overexpressing cells compared to normal cells. Our data suggests that the increase in P-cadherin seen in early oral cancer may promote tumor aggressiveness by altering cell behavior, and increasing EMT- related signaling. The contribution of P-cadherin to tumor development may be helpful in identifying biological targets for oral cancer therapies in dysplastic tissue. Citation Format: Desseree Lysne, Michelle Perkins, James Johns, Matthew Van Dam, Darren Cuevas, Kathryn Lawson. P-Cadherin overexpression modulates insulin-like growth factor signaling in oral squamous carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3989. doi:10.1158/1538-7445.AM2013-3989

Abstract 3044: Prognostic impact of P-cadherin overexpression associated with CDH3 promoter hypomethylation in intrahepatic cholangiocarcinoma (ICC).

Abstract Background. P-cadherin belongs to the family of classic cadherin, and accumulating evidences shows that P-cadherin is strongly associated with carcinogenesis and confers malignant phenotype of various cancers including breast, pancreatic, and colon carcinomas. Intrahepatic cholangiocarcinoma (ICC) is the second most common subtype of primary hepatobillary cancer. Despite advances in therapy, its survival rate remains poor. This study aimed to analyze P-cadherin expression and its prognostic value in Intrahepatic cholangiocarcinoma and to validate the P-cadherin gene (CDH3) promoter methylation status as the mechanism of this gene regulation. Methods. Fifty-nine patients diagnosed with ICC who underwent hepatic resection in our department were enrolled in this study. Using the paraffin-embedded tumor tissues, expression status of P-cadherin was evaluated and correlated with various clinicopathological features (age, gender, tumor markers, tumor diameter and number, stage, gross type, grade of tumor differentiation, vessel invasion, and lymph node metastases) and prognosis. Furthermore, P-cadherin expression and CDH3 promoter methylation status in 8 cholangiocarcinoma cell lines (HuCCT1, YSCCC, MEC, OZ, HuH28, RBE, SSP25, and TKKK) were analyzed. Results. In ICC tissues, 50.8% (30/59) of cases shows a high expression of P-cadherin by immunohistochemical analysis, and high expression of P-cadherin was significantly correlated with tumor diameter > 3.8 cm (p=0.0006). In multivariate analyses, P-cadherin expression was the independent prognostic factors for disease-free survival (relative risk:2.72, p=0.0138) and overall survival (relative risk:3.23 p=0.0012). Of the 8 cell lines, 3 (HuCCT1, YSCCC, OZ) were found to express P-cadherin protein and mRNA. These cell lines showed complete CDH3 promoter unmethylation. However, other cell lines showed partial methylation and unmetylation. From this result, the CDH3 promoter methylation was found to be related to the expression of P-cadherin. Conclusion. These results suggested that expression of P-cadherin is a useful prognostic marker and seems to be important in the malignant behavior in ICC. We are now conducting experiments to validate the CDH3 methylation status in the cancerous tissues and their adjacent normal counterpart, and investigating the biological role of P-cadherin in ICC. Citation Format: Keita Sakamoto, Katsunori Imai, Shigeki Nakagawa, Hirohisa Okabe, Hidetoshi Nitta, Hiromitsu Hayashi, Akira Chikamoto, Takatoshi Ishiko, Toru Beppu, Hideo Baba. Prognostic impact of P-cadherin overexpression associated with CDH3 promoter hypomethylation in intrahepatic cholangiocarcinoma (ICC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3044. doi:10.1158/1538-7445.AM2013-3044