PAR Genes: Molecular Probes to Pathological Assessment in Breast Cancer Progression

Taking the issue of tumor categorization a step forward and establish molecular imprints to accompany histopathological assessment is a challenging task. This is important since often patients with similar clinical and pathological tumors may respond differently to a given treatment. Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the first member of the mammalian PAR family consisting of four genes. PAR1 and PAR2 play a central role in breast cancer. The release of N-terminal peptides during activation and the exposure of a cryptic internal ligand in PARs, endow these receptors with the opportunity to serve as a “mirror-image” index reflecting the level of cell surface PAR1&2-in body fluids. It is possible to use the levels of PAR-released peptide in patients and accordingly determine the choice of treatment. We have both identified PAR1 C-tail as a scaffold site for the immobilization of signaling partners, and the critical minimal binding site. This binding region may be used for future therapeutic modalities in breast cancer, since abrogation of the binding inhibits PAR1 induced breast cancer. Altogether, both PAR1 and PAR2 may serve as molecular probes for breast cancer diagnosis and valuable targets for therapy

Regulation of human protease-activated receptor 1 (hPar1) gene expression in breast cancer by estrogen

A pivotal role is attributed to the estrogenreceptor (ER) pathway in mediating the effect of estrogen in breast cancer progression. Yet the precise mechanisms of cancer development by estrogen remain poorly understood. Advancing tumor categorization a step forward, and identifying cellular gene fingerprints to accompany histopathological assessment may provide targets for therapy as well as vehicles for evaluating the response to treatment. We report here that in breast carcinoma, estrogen may induce tumor development by eliciting protease-activated receptor-1 (PAR1) gene expression. Induction of PAR1 was shown by electrophoretic mobility shift assay, luciferase reporter gene driven by the hPar1 promoter, and chromatin-immunoprecipitation analyses. Functional estrogen regulation of hPar1 in breast cancer was demonstrated by an endothelial tube-forming network. Notably, tissue-microarray analyses from an established cohort of women diagnosed with invasive breast carcinoma exhibited a significantly shorter disease-free (P 0.006) and overall (P 0.02) survival of patients that were positive for ER and PAR1, compared to ER-positive but PAR1-negative patients. We propose that estrogen transcriptionally regulates hPar1, culminating in an aggressive gene imprint in breast cancer. While ER patients are traditionally treated with hormone therapy, the presence of PAR1 identifies a group of patients that requires additional treatment, such as anti-PAR1 biological vehicles or chemotherapy.—Salah, Z., Uziely, B., Jaber, M., Maoz, M., Cohen, I., Hamburger, T., Maly, B., Peretz, T., B.-S, R. Regulation of human protease-activated receptor 1 (hPar1) gene expression in breast cancer by estrogen

Etk/Bmx Regulates Proteinase-Activated-Receptor1 (PAR1) in Breast Cancer Invasion: Signaling Partners, Hierarchy and Physiological Significance

BACKGROUND: While protease-activated-receptor 1 (PAR(1)) plays a central role in tumor progression, little is known about the cell signaling involved. METHODOLOGY/PRINCIPAL FINDINGS: We show here the impact of PAR(1) cellular activities using both an orthotopic mouse mammary xenograft and a colorectal-liver metastasis model in vivo, with biochemical analyses in vitro. Large and highly vascularized tumors were generated by cells over-expressing wt hPar1, Y397Z hPar1, with persistent signaling, or Y381A hPar1 mutant constructs. In contrast, cells over-expressing the truncated form of hPar1, which lacks the cytoplasmic tail, developed small or no tumors, similar to cells expressing empty vector or control untreated cells. Antibody array membranes revealed essential hPar1 partners including Etk/Bmx and Shc. PAR(1) activation induces Etk/Bmx and Shc binding to the receptor C-tail to form a complex. Y/A mutations in the PAR(1) C-tail did not prevent Shc-PAR(1) association, but enhanced the number of liver metastases compared with the already increased metastases obtained with wt hPar1. We found that Etk/Bmx first binds via the PH domain to a region of seven residues, located between C378-S384 in PAR(1) C-tail, enabling subsequent Shc association. Importantly, expression of the hPar1-7A mutant form (substituted A, residues 378-384), which is incapable of binding Etk/Bmx, resulted in inhibition of invasion through Matrigel-coated membranes. Similarly, knocking down Etk/Bmx inhibited PAR(1)-induced MDA-MB-435 cell migration. In addition, intact spheroid morphogenesis of MCF10A cells is markedly disrupted by the ectopic expression of wt hPar1. In contrast, the forced expression of the hPar1-7A mutant results in normal ball-shaped spheroids. Thus, by preventing binding of Etk/Bmx to PAR(1) -C-tail, hPar1 oncogenic properties are abrogated. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration that a cytoplasmic portion of the PAR(1) C-tail functions as a scaffold site. We identify here essential signaling partners, determine the hierarchy of binding and provide a platform for therapeutic vehicles via definition of the critical PAR(1)-associating region in the breast cancer signaling niche

Cytologic diagnosis of meningioma metastatic to the pleural cavity

Intracranial meningiomas seldom locally recur after surgical resection. Extracranial metastases of meningioma are more uncommon, and the lung is the most common site of metastasis.1,9 Metastases to the pleura are extremely rare. We report a case of metastatic meningioma diagnosed by cytologic examination of pleural fluid.7 A 60-year-old woman presented with increasing shortness of breath of a few weeks\u92 duration. Chest radiography revealed a massive right pleural effusion. Thoracic computed tomography (CT) after pleural fluid evacuation showed right pleural nodularity and multiple bilateral pulmonary nodules. The patient had had a meningioma surgically removed in 1986. In 1988, a follow-up CT scan showed recurrent tumor even though the patient had no clinical signs of illness. Complete surgical removal of this mass was performed. The tumor recurred after 4 years and was surgically excised; the patient was treated with cranial radiotherapy and chemotherapy. She remained well until 2000, when respiratory problems developed. A CT scan at that time did not disclose an intracranial tumor. Pleural fluid was sent for cytologic examination. The smears contained numerous cells arranged singly and in small clusters, often creating meningotheliomatous whorls. (Figure 1). Neoplastic cells were somewhat pleomorphic, with round nuclei, granular cytoplasm and an epithelioid appearance. Nuclei of tumor cells varied slightly in size, nuclear chromatin showed a rather finely granular pattern, and nucleoli were small and inconspicuous. Occasional cytoplasmic invagination into nuclei (nuclear pseudoinclusions) was observed. No mitotic figures were found. After the diagnosis of metastatic meningioma, fine needle aspiration biopsy (FNAB) of the largest pleural and pulmonary nodules was performed under CT guidance. The histologic findings for both nodules were similar. The sections showed sheets of neoplastic cells with some round but mostly spindle-shaped, elongated or fusiform nuclei, sometimes arranged in parallel formations. Occasionally the tumor cells had indistinct cell borders and round or ovoid nuclei and formed syncytial sheets in which a few whorl formations were observed (Figure 2A and B). The nuclei of the tumor cells were slightly pleomorphic, and the abundant cytoplasm varied in size and shape. Nuclear chromatin showed a finely granular pattern. Rare intranuclear cytoplasmic pseudoinclusions and mitoses were seen. No psammoma bodies were found. The diagnosis was supported by positive immunohistochemical reactions for epithelial membrane antigen and vimentin and negative ones for keratin, calretinin, S-100 protein and MOC-1. Proliferating cell nuclear antigen (MIB-1 monoclonal staining of Ki-67 nuclear protein) was positive in 10% of nuclei. The histology of the primary intracranial tumor, resected in 1986, and of the recurrent tumors, resected in 1988 and 1992, was similar and had a syncytial and fibroblastic appearance. No necrosis was present. No evidence of infiltration of brain tissue was found, but in the recurrent tumors there were signs of cellular atypia, increased mitotic activity (Figure 3) and tumor penetration of bone. The patient was treated with chemotherapy and was well at this writing. The World Health Organization currently recognizes 3 grades of meningioma: grade 1 (benign), grade 2 (atypical) and grade 3 (anaplastic or malignant). The histologic criteria used to define atypical and malignant tumors include loss of architectural pattern, necrosis, prominent nucleoli, nuclear pleomorphism, high cellularity, high mitotic rate and high nuclear/ cytoplasmic ratios.2,4 Atypical meningioma is quite rare, accounting for 6% of primary intracranial meningiomas, but displays a significant 10-year recurrence rate of 66% as compared with 2% for benign meningioma.3,6 The interval to recurrence may be several years after initial surgery.4 The risk of metastasis is further clouded by possible iatrogenic spread of the neoplasm through seeding of the venous sinuses during craniotomy.8 Most metastatic meningiomas are derived from meningiomas that had been previously excised and had at least atypical histologic features. High MIB-1 labeling proliferation index is usually related to atypical and malignant histology, with aggressive clinical behavior. However, the prognosis cannot be based on the results of high-level MIB-1 studies only since how high is too high remains unclear.3,5 The latency interval between the time of initial diagnosis of the intracranial lesion and discovery of metastasis varies from a few months to 18 years.8 The lungs are the most common sites of metastatic meningiomas, but pleural metastases are very rare. For a cytologic diagnosis of metastatic meningiomas, other primary and metastatic neoplasms, such as malignant mesothelioma, metastatic adenocarcinoma, squamous cell carcinoma and melanoma, should be included in the differential diagnosis. The most common neoplasms to metastasize to the pleura are carcinomas from the lung, breast and ovary. Also, meningioma metastatic to the pleural cavity, should be considered in the interpretation of a pleural effusion in patient with a history of recurrent intracranial meningioma. A careful study of the history, histopathologic correlation with previous resections and immunohistologic profile are tools for reach the correct diagnosi

Isolated carcinoid tumor metastatic to the thyroid gland: report of a case initially diagnosed by fine needle aspiration cytology

Neuroendocrine tumor metastatic to the thyroid gland is rare and may be difficult to differentiate from primary thyroid neuroendocrine tumors, such as medullary thyroid carcinoma (MTC). This report describes an unusual case of bronchial carcinoid metastatic to the thyroid diagnosed by fine needle aspiration cytology (FNAC). Case A 42-year-old woman with an undiagnosed bronchial carcinoid tumor presented to our clinic with a solitary nodule in the thyroid gland. FNAC of the nodule showed loosely cohesive groups of cuboidal tumor cells with scant, slightly granular cytoplasm; centrally located nuclei with a coarsely granular, salt-and-pepper chromatin pattern and inconspicuous nucleoli. Immunocytochemically the tumor cells were positive for neuron-specific enolase, chromogranin and synaptophysin and negative for thyroglobulin, calcitonin and carcinoembryonic antigen. The cytologic diagnosis of a metastatic neuroendocrine carcinoma was confirmed histologically. Conclusion: Metastasis to the thyroid gland may pose a diagnostic problem, particularly with tumors of neuroendocrine origin, as these have similar cytologic features in various organs. The correct preoperative cytologic diagnosis of metastatic carcinoid tumor in patients without a prior history of cancer and differential diagnosis with MTC are crucial because prognosis, workup and treatment are different in eac

Nodular fasciitis of the breast: Report of a case initially diagnosed by fine needle aspiration cytology

BACKGROUND: Nodular fasciitis is a rare benign pseudosarcomatous proliferation of fibroblasts in the breast, in which the clinical examination and mammographic findings may closely mimic mammary carcinoma. CASE: A case of nodular fasciitis was diagnosed by fine needle aspiration. A 15-year-old girl was admitted to our hospital with a recently noticed, rapidly growing mass in the right breast. The aspirate contained cohesive groups of fusiform cells with elongated and oval nuclei, regular nuclear membranes and inconspicuous nucleoli, intermingled with scattered lymphocytes, red blood cells and characteristic granular background substance. A cytologic diagnosis of nodular fasciitis was made and confirmed histologically. CONCLUSION: Mammary nodular fasciitis is often clinically suspicious for carcinoma and rarely diagnosed by fine needle aspiration cytology. A literature search yielded only five reported cases. The cytologic diagnosis of this entity helps to choose the correct surgical procedure, preventing psychological trauma to the patient

Heparanase Accelerates Obesity-Associated Breast Cancer Progression

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RNF20 and histone H2B ubiquitylation exert opposing effects in Basal-Like versus luminal breast cancer

Breast cancer subtypes display distinct biological traits that influence their clinical behavior and response to therapy. Recent studies have highlighted the importance of chromatin structure regulators in tumorigenesis. The RNF20-RNF40 E3 ubiquitin ligase complex monoubiquitylates histone H2B to generate H2Bub1, while the deubiquitinase (DUB) USP44 can remove this modification. We found that RNF20 and RNF40 expression and global H2Bub1 are relatively low, and USP44 expression is relatively high, in basal-like breast tumors compared with luminal tumors. Consistent with a tumor-suppressive role, silencing of RNF20 in basal-like breast cancer cells increased their proliferation and migration, and their tumorigenicity and metastatic capacity, partly through upregulation of inflammatory cytokines. In contrast, in luminal breast cancer cells, RNF20 silencing reduced proliferation, migration and tumorigenic and metastatic capacity, and compromised estrogen receptor transcriptional activity, indicating a tumor-promoting role. Notably, the effects of USP44 silencing on proliferation and migration in both cancer subtypes were opposite to those of RNF20 silencing. Hence, RNF20 and H2Bub1 have contrasting roles in distinct breast cancer subtypes, through differential regulation of key transcriptional programs underpinning the distinctive traits of each subtype

Macrophages Upregulate Estrogen Receptor Expression in the Model of Obesity-Associated Breast Carcinoma

Breast cancer (BC) and obesity are two heterogeneous conditions with a tremendous impact on health. BC is the most commonly diagnosed neoplasm and the leading cause of cancer-related mortality among women, and the prevalence of obesity in women worldwide reaches pandemic proportions. Obesity is a significant risk factor for both incidence and worse prognosis in estrogen receptor positive (ER+) BC. Yet, the mechanisms underlying the association between excess adiposity and increased risk/therapy resistance/poorer outcome of ER+, but not ER−negative (ER−), BC are not fully understood. Tumor-promoting action of obesity, predominantly in ER + BC patients, is often attributed to the augmented production of estrogen in ‘obese’ adipose tissue. However, in addition to the estrogen production, expression levels of ER represent a key determinant in hormone-driven breast tumorigenesis and therapy response. Here, utilizing in vitro and in vivo models of BC, we show that macrophages, whose adverse activation by obesogenic substances is fueled by heparanase (extracellular matrix-degrading enzyme), are capable of upregulating ER expression in tumor cells, in the setting of obesity-associated BC. These findings underscore a previously unknown mechanism through which interplay between cellular/extracellular elements of obesity-associated BC microenvironment influences estrogen sensitivity—a critical component in hormone-related cancer progression and resistance to therapy