Francesco Caiazza

Role of ERβ palmitoylation in the inhibition of human colon cancer cell proliferation

The cellular functions regulated by 17b-estradiol (E2) start after the hormone binds to its receptors (i.e., ERa and ERb). These act as ligand-dependent transcription factor transactivating target genes. In addition, E2 induces non-genomic actions, whose activation is triggered by a fraction of the ERs localized at the plasma membrane. Palmitoylation allows ERa to localize at the plasma membrane, to associate with caveolin-1, and, upon E2 stimulation, to activate rapid signals relevant for cell proliferation. The existence of a mechanism, which allows ERb localization at the plasma membrane and its putative role in anti-proliferative E2 effects is completely unknown. Here, the susceptibility of ERb to undergo palmitoylation and the role played by this process has been analyzed in DLD-1 containing endogenous ERb or in HeLa cells transiently transfected with ERb or ERa expression vectors. As for ERa, palmitoylation is necessary for ERb localization at the plasma membrane and its association with caveolin-1 but, in contrast to ERa, the E2 binding increases ERb association with caveolin-1 and the p38 member of MAPK family. Moreover, the palmitoyl acyl transferase (PAT) inhibitor blocks the ability of ERb–E2 complex to activate p38 impairing the receptor-dependent activation of downstream pro-apoptotic cascade (i.e., caspase3 activation and poly(ADP-ribose)polymerase (PARP) cleavage). Consequently, palmitoylation must be considered to be a molecular device for ERb, which allows these receptors to interact with the plasma membrane and to regulate E2-induced non-genomic functions relevant to the antiproliferative effect of this hormone.

Estrogen Receptors and Their Implications in Colorectal Carcinogenesis

Upon binding their cognate receptors, ERα (ESR1) and ERβ (ESR2), estrogens activate intracellular signaling cascades that have important consequences for cellular behavior. Historically linked to carcinogenesis in reproductive organs, estrogens have also been implicated in the pathogenesis of different cancer types of non-reproductive tissues including the colon. ERβ is the predominant estrogen receptor expressed in both normal and malignant colonic epithelium. However, during colon cancer progression, ERβ expression is lost, suggesting that estrogen signaling may play a role in disease progression. Estrogens may in fact exert an anti-tumor effect through selective activation of pro-apoptotic signaling mediated by ERβ, inhibition of inflammatory signals and modulation of the tumor microenvironment. In this review, we analyze the estrogen pathway as a possible therapeutic avenue in colorectal cancer, we report the most recent experimental evidence to explain the cellular and molecular mechanisms of estrogen-mediated protection against colorectal tumorigenesis, and we discuss future challenges and potential avenues for targeted therapy.

ADAM-17: a novel therapeutic target for triple negative breast cancer

BACKGROUND Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors. PATIENTS AND METHODS Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs. RESULTS In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents. CONCLUSION It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.BACKGROUND Validated targeted therapy is currently unavailable for patients with invasive breast cancer negative for oestrogen receptors, progesterone receptors and HER2 [i.e., those with triple-negative (TN) disease]. ADAM-17 is a protease involved in the activations of several ligands that bind to and promotes intracellular signalling from the EGFR/HER family of receptors. PATIENTS AND METHODS Expression of ADAM-17 was measured in 86 triple-negative and 96 non-triple-negative breast cancers. The ADAM-17 specific inhibitor, PF-5480090 (TMI-002, Pfizer) was tested in a panel of breast cancer cell lines for effects on functional outputs. RESULTS In this study we show using both Western blotting and immunohistochemistry that ADAM-17 is expressed at significantly higher levels in TN than non-TN breast cancers. Using a panel of breast cancer cell lines in culture, PF-5480090 was found to decrease release of the EGFR ligand, TGF-alpha, decrease levels of phosphorylated EGFR and block cell proliferation in a cell-type-dependent manner. Potentially important was the finding of a significant and moderately strong correlation between ADAM-17 activity and extent of proliferation inhibition by PF-5480090 (r = 0.809; p = 0.003; n = 11). Pretreatment of cell lines with PF-5480090 enhanced response to several different cytotoxic and anti-EGFR/HER agents. CONCLUSION It is concluded that inhibition of ADAM-17, especially in combination with chemotherapy or anti-EGFR/HER inhibitors, may be a new approach for treating breast cancer, including patients with TN disease.

Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells.

Background:Identification and validation of a targeted therapy for triple-negative breast cancer (TNBC), that is, breast cancers negative for oestrogen receptors, progesterone receptors and HER2 amplification, is currently one of the most urgent problems in breast cancer treatment. EGFR is one of the best-validated driver genes for TNBC. EGFR is normally activated following the release of ligands such as TGFα, mediated by the two MMP-like proteases ADAM (a disintegrin and metalloproteinase)-10 and ADAM-17. The aim of this study was to investigate the antitumour effects of a monoclonal antibody against ADAM-17 on an in vitro model of TNBC.Methods:We investigated an inhibitory cross-domain humanised monoclonal antibody targeting both the catalytic domain and the cysteine-rich domain of ADAM17-D1(A12) in the HCC1937 and HCC1143 cell lines.Results:D1(A12) was found to significantly inhibit the release of TGFα, and to decrease downstream EGFR-dependent cell signalling. D1(A12) treatment reduced proliferation in two-dimensional clonogenic assays, as well as growth in three-dimensional culture. Furthermore, D1(A12) reduced invasion of HCC1937 cells and decreased migration of HCC1143 cells. Finally, D1(A12) enhanced cell death in HCC1143 cells.Conclusion:Our in vitro findings suggest that targeting ADAM-17 with D1(A12) may have anticancer activity in TNBC cells.

Cytosolic phospholipase A2 activation correlates with HER2 overexpression and mediates estrogen-dependent breast cancer cell growth.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) catalyzes the hydrolysis of membrane glycerol-phospholipids to release arachidonic acid as the first step of the eicosanoid signaling pathway. This pathway contributes to proliferation in breast cancer, and numerous studies have demonstrated a crucial role of cyclooxygenase 2 and prostaglandin E(2) release in breast cancer progression. The role of cPLA(2)alpha activation is less clear, and we recently showed that 17beta-estradiol (E2) can rapidly activate cPLA(2)alpha in MCF-7 breast cancer cells. Overexpression or gene amplification of HER2 is found in approximately 30% of breast cancer patients and correlates with a poor clinical outcome and resistance to endocrine therapy. This study reports the first evidence for a correlation between cPLA(2)alpha enzymatic activity and overexpression of the HER2 receptor. The activation of cPLA(2)alpha in response to E2 treatment was biphasic with the first phase dependent on trans-activation through the matrix metalloproteinase-dependent release of heparin-bound epidermal growth factor. EGFR/HER2 heterodimerization resulted in downstream signaling through the ERK1/2 cascade to promote cPLA(2)alpha phosphorylation at Ser505. There was a correlation between HER2 and cPLA(2)alpha expression in six breast cancer cell lines examined, and inhibition of HER2 activation or expression in the SKBR3 cell line using herceptin or HER2-specific small interfering RNA, respectively, resulted in decreased activation and expression of cPLA(2)alpha. Pharmacological blockade of cPLA(2)alpha using a specific antagonist suppressed the growth of both MCF-7 and SKBR3 cells by reducing E2-induced proliferation and by stimulating cellular apoptosis and necrosis. This study highlights cPLAalpha(2) as a potential target for therapeutic intervention in endocrine-dependent and endocrine-independent breast cancer.

Preclinical evaluation of the AR inhibitor enzalutamide in triple-negative breast cancer cells.

The androgen receptor (AR) is present in approximately 80% of invasive breast cancer patients and in up to 30% of patients with triple-negative breast cancer (TNBC). Therefore, our aim was to investigate the targeting of AR as a possible hormonal approach to the treatment of TNBC. Analysis of 2091 patients revealed an association between AR expression and poor overall survival, selectively in patients with the basal subtype of breast cancer, the vast majority of which are TNBC. IC50 values for the second-generation anti-androgen enzalutamide across 11 breast cancer cell lines varied from 4 µM to >50 µM. The activity of enzalutamide was similar in TN and non-TN cell lines but was dependent on the presence of AR. Enzalutamide reduced clonogenic potential and cell growth in a 3D matrix in AR-positive cells. In addition, enzalutamide also inhibited cell migration and invasion in an AR-dependent manner. Enzalutamide appeared to mediate these processes through down-regulation of the transcription factors AP-1 and SP-1. The first-generation anti-androgen flutamide similarly blocked cell growth, migration and invasion. AR-positive TNBC cells clustered separately from AR-negative cells based on an androgen-related gene expression signature, independently of TNBC subtype. We conclude that targeting of the AR with drugs such as enzalutamide may provide an alternative treatment strategy for patients with AR-positive TNBC.

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours

Background:The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A2α (cPLA2α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA2α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines.Methods:The importance of differential cPLA2α activity in clinical breast cancer was established by relating the expression of cPLA2α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters.Results:High cPLA2α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA2α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA2α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up.Conclusion:This study shows a role of cPLA2α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.

Estrogen, phospholipase A and breast cancer.

The development of breast cancer is promoted by diverse factors that impact on intracellular signaling to promote proliferation and cell survival. The role of eicosanoid signaling through prostaglandin release and the up-regulation of cyclooxygenase-2 (COX-2) is established, however, the impact of phospholipase A (PLA) activity and over-expression is less certain. Here we review current literature concerning the role of PLA in breast cancer and describe how eicosanoid signaling may be a facet of estrogen-stimulated breast cancer etiology and progression.

Targeting EGFR in metastatic colorectal cancer beyond the limitations of KRAS status: alternative biomarkers and therapeutic strategies.

Patients with metastatic colorectal cancer have a very poor prognosis. Incorporation of targeted molecular therapies, such as the anti-EGFR receptor monoclonal antibodies cetuximab and panitumumab, into treatment regimens has improved outcomes for patients with wild-type RAS tumors. Yet, response rates remain low and overall survival times are short. Increased understanding of oncogenic signaling pathways within the tumor, and how these are regulated by the inflammatory tumor microenvironment, is a priority to facilitate the development of biomarkers to better guide the use of existing therapies and to develop new ones. Here, we review recent preclinical and clinical progress in the development of biomarkers for predicting response to anti-EGFR therapy in metastatic colorectal cancer.

Oocyte Maturation: A story of arrest and release

The release of a mature healthy egg for fertilization is the center of the entire reproductive process. From the time of embryonic development till fertilization, the oocyte undergoes several stop-and-go periods. In most animals, oocytes are held in meiotic arrest in prophase I prior to ovulation. The ovulatory luteinizing hormone (LH) surge promotes the resumption of meiosis of the arrested oocytes and their progression through the second meiotic cycle, only to be arrested again at metaphase II until fertilization. This review addresses the underlying mechanisms involved in maintaining the oocyte in meiotic arrest as well as the signaling pathways responsible for releasing it from the arrested phase just prior to ovulation until the completion of meiosis at the time of fertilization.