Mitsuko Furuya

Expression of regulator of G protein signalling protein 5 (RGS5) in the tumour vasculature of human renal cell carcinoma

The gene expression profiles of tumour and normal vasculature are distinctively different. The altered expression of various angiogenesis‐related genes in tumour‐derived endothelial cells has been investigated intensively, but there may be as yet unidentified molecules that regulate tumour angiogenesis. In the present study, the distinctive expression of regulator of G protein signalling protein 5 (RGS5) in tumour vessels in human renal cell carcinoma (RCC) has been clarified. RGS5 is a member of the RGS superfamily and acts as a negative regulator of heterotrimeric G protein‐mediated signalling through G protein‐coupled receptors (GPCRs). RT‐PCR showed strong expression of RGS5 in all RCCs examined, but expression was very weak or undetectable in normal kidneys. By real‐time RT‐PCR, the ratio of RGS5 mRNA in RCC to that in normal kidney was 6.6 : 1 (p = 0.0012). In situ hybridization showed strong expression of RGS5 in vessels within tumour cell nests. It was expressed neither in tumour cells nor in normal renal capillaries. Immunohistochemical staining using serial sections for endothelial cell markers (CD31 and CD34) and smooth muscle cell markers (α‐SMA and desmin), as well as fluorescence double staining, strongly suggested that tumour endothelial cells were the main location of RGS5 in RCC. These findings suggest that RGS5 may be involved in G protein‐mediated signalling in tumour vessels in human RCC. Copyright

Expression of an activated mammalian target of rapamycin (mTOR) in gastroenteropancreatic neuroendocrine tumors

AimsGastroenteropancreatic neuroendocrine tumors are rare, and the current WHO classification divides this tumor entity into well-differentiated (neuro)endocrine tumors, well-differentiated (neuro)endocrine carcinomas, and poorly differentiated (neuro)endocrine carcinomas. Poorly differentiated (neuro)endocrine carcinoma is extremely aggressive, and no appropriate therapeutic approach has been established. The mammalian target of rapamycin (mTOR), an important regulator of cell proliferation and protein translation, is activated in various malignancies. Recent phase II trial has revealed the efficacy of mTOR inhibitor (RAD001; everolimus) against low-to-intermediate grade neuroendocrine tumors. However, the beneficial role of mTOR inhibitor against poorly neuroendocrine carcinoma remains uncertain. The purpose of the present study was to determine the activation of mTOR in gastropancreatic neuroendocrine tumors, especially in poorly differentiated neuroendocrine carcinomas.MethodsExpression of p-mTOR(Ser2448) was assessed by immunohistochemistry in 20 gastropancreatic neuroendocrine tumors (seven well-differentiated neuroendocrine tumors, four well-differentiated neuroendocrine carcinomas, and nine poorly differentiated neuroendocrine carcinomas). Double immunohistochemistry was performed with p-Akt for patients with high p-mTOR expression.ResultsExpression of mTOR was seen in 9 (45%) of 20 gastroenteropancreatic neuroendocrine tumors. High expression of p-mTOR was seen in 6 (67%) of 9 poorly differentiated neuroendocrine carcinomas which was higher than the expression rate of well-differentiated neuroendocrine tumors and carcinomas, 3 (27%) of 11. All large cell neuroendocrine carcinomas showed high p-mTOR expression. Some tumor cells showed positive staining for p-mTOR co-expressed p-Akt.ConclusionsHigh expression rate of p-mTOR in poorly differentiated neuroendocrine carcinomas (large-cell type) may suggest the potential role of mTOR inhibitors as effective therapeutic agents for this highly malignant disease.

Pulmonary cysts of Birt-Hogg-Dubé syndrome: a clinicopathologic and immunohistochemical study of 9 families.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by fibrofolliculomas, renal tumors, and pulmonary cysts with recurrent pneumothorax. Multiple pulmonary cysts and pneumothorax are the key signs for diagnosing BHD syndrome. The pathologic features of BHD pulmonary cysts, however, are poorly understood. This disorder is caused by mutations in the gene that encodes folliculin (FLCN). FLCN is regarded as a tumor suppressor; it mediates cellular activities by interacting with the mammalian target of rapamycin (mTOR). In this study, we investigated the lungs of 11 patients from 9 BHD families. The majority of patients consulting doctors were women between 30 and 60 years of age who had pulmonary cysts and repeated pneumothoraces. Genomic DNA testing revealed 5 different mutation patterns. Histopathologic examination found that the inner surface of cysts was lined by epithelial cells, sometimes with a predominance of type II pneumocyte-like cuboidal cells. The cysts occasionally contained internal septa consisting of alveolar walls or showed an “alveoli within an alveolus” pattern. The cells constituting the cysts stained positive for phospho-S6 ribosomal protein expression, suggesting activation of the mTOR pathway. Although BHD pulmonary cysts are frequently misdiagnosed as nonspecific cystic diseases, they are distinctly different in histopathology from other bullous changes. Mechanical stress such as rupture and postrupture remodeling allows mesothelial invagination and fibrosis. Such modified BHD pulmonary cysts are virtually indistinguishable from nonspecific blebs and bullae. We propose a new insight, namely, that the BHD syndrome-associated pulmonary cyst may be considered a hamartoma-like cystic alveolar formation associated with deranged mTOR signaling.

Down-regulation of CD9 in Human Ovarian Carcinoma Cell Might Contribute to Peritoneal Dissemination: Morphologic Alteration and Reduced Expression of β1 Integrin Subsets

Peritoneal dissemination is one of the main causes of death in cancer patients. Pathophysiology of metastasis has been well investigated, but the mechanism of diffuse spread of tumor colonies in the peritoneal cavity is not fully understood. CD9 is a member of tetraspanin and its down-regulation is known to be involved in poor prognosis. To investigate the significance of the down-regulation of CD9, HTOA, an ovarian carcinoma cell line that highly expressed CD9, was transiently transfected with small interfering RNA (siRNA) against CD9, and CD9-negative cells (HTOA(CD9-)) were purified. HTOA(CD9-) showed altered adhesion patterns on Matrigel, collagen, fibronectin, and laminin compared with those of control siRNA-transfected HTOA (control-HTOA). Flow cytometry and fluorescence cytostainings revealed that the expression levels of integrins beta1, alpha2, alpha3beta1, alpha5, and alpha6 were lower in HTOA(CD9-) than those of control-HTOA. HTOA(CD9-) showed altered expression of junctional and cytoskeletal molecules. By time-lapse video microscopy, control-HTOA showed solid adhesion to extracellular matrix and formed cobblestone pattern, whereas HTOA(CD9-) showed weaker adhesion and were distributed as diffuse spots. To examine whether the expression level of CD9 change during tumor dissemination, HTOA-P, a highly disseminative subclone of HTOA, was established. HTOA-P showed distinctive down-regulation of CD9 at mRNA and protein levels, and showed similar morphologic alteration as HTOA(CD9-) did. These findings indicate that the down-regulation of CD9 may be an acquired event in the process of tumor dissemination. Down-regulated CD9 may attenuate the expression of several integrins and rearrange junctional and cytoskeletal molecules that might contribute to dissemination of ovarian carcinomas.

Up‐regulation of the interferon γ (IFN‐γ)‐inducible chemokines IFN‐inducible T‐cell α chemoattractant and monokine induced by IFN‐γ and of their receptor CXC receptor 3 in human renal cell carcinoma

The antiangiogenic CXC chemokines interferon γ (IFN‐γ)‐inducible T‐cell α chemoattractant (I‐TAC) and monokine induced by IFN‐γ (Mig) are known as members of IFN‐γ‐inducible antiangiogenic CXC chemokines. However, the expression of these chemokines in highly angiogenic tumors remains poorly understood. The authors examined expression of I‐TAC, Mig, and their receptor, CXCR3, in tissue samples from patients with renal cell carcinoma (RCC).

CD151 dynamics in carcinoma-stroma interaction: integrin expression, adhesion strength and proteolytic activity.

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and α3β1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5CD151−), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5CD151−, striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. α3β1 integrin was internalized in part, and α6β4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5CD151−. Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma–stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.

Birt–Hogg–Dubé syndrome: clinicopathological features of the lung

Birt–Hogg–Dubé syndrome (BHD) is an autosomal dominant inherited disorder characterised by fibrofolliculomas, renal tumours, pulmonary cysts and pneumothorax. The pulmonary cysts and repeated episodes of pneumothorax are the clinical hallmarks for discovering families affected by the syndrome. This disorder is caused by mutations in the gene coding for folliculin (FLCN). FLCN forms a complex with FLCN-interacting protein 1 (FNIP1) and FNIP2 (also known as FNIPL), and the complex cross-talks with signalling molecules such as 5′-AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR). Heterozygous Flcn knockout mice and rats with Flcn gene mutations develop renal cysts, adenomas and/or carcinomas. These findings suggest that FLCN functions as a tumour suppressor that inhibits renal carcinogenesis. However, the mechanisms of the formation of pulmonary cysts and pneumothorax associated with heterozygous mutations in FLCN are poorly understood. Resected lung specimens from patients with BHD are often misdiagnosed by pathologists as non-specific blebs or bullae or emphysema, and patients with BHD who have pulmonary cysts and repeated pneumothorax frequently do not receive appropriate medical investigations. This review discusses the clinical and pathological features of lungs of patients with BHD, focusing on the diagnostic pathology and possible mechanisms of cyst formation.

Pathophysiology of placentation abnormalities in pregnancy-induced hypertension

During embryogenesis and development, the fetus obtains oxygen and nutrients from the mother through placental microcirculation. The placenta is a distinctive organ that develops and differentiates per se, and that organizes fetal growth and maternal condition in the entire course of gestation. Several life-threatening diseases during pregnancy, such as pregnancy-induced hypertension (PIH) and eclampsia, are closely associated with placental dysfunction. Genetic susceptibilities and poor placentation have been investigated intensively to understand the pathophysiology of PIH. It is currently thought that “poor placentation hypothesis”, in which extravillous trophoblasts fail to invade sufficiently the placental bed, explains in part maternal predisposition to this disease. Cumulative studies have suggested that hypoxic micromilieu of fetoplacental site, shear stress of uteroplacental blood flow, and aberrantly secreted proinflammatory substances into maternal circulation synergistically contribute to the progression of PIH. For example, soluble form of vascular endothelial growth factor receptor-1 (sVEGFR-1) and soluble form of CD105 are elevated in circulation of PIH mothers. However, it remains to be poorly understood the pathological events in the placenta during the last half of gestation as maternal systemic disorders get worse. For better understanding and effective therapeutic approaches to PIH, it is important to clarify pathological course of PIH-associated changes in the placenta. In this review, current understanding of placental development and the pathophysiology of PIH placenta are summarized. In addition, recent findings of vasoactive signalings in PIH and rodent PIH models are discussed.

Lung cysts in Birt-Hogg-Dube syndrome : Histopathological characteristics and aberrant sequence repeats

Birt‐Hogg‐Dubé (BHD) syndrome is a rare disorder inherited in an autosomal dominant manner. The affected patients are predisposed to cutaneous fibrofolliculomas, renal cell tumors and lung cysts with recurrent pneumothorax. Contrary to neoplastic events in the skin and the kidney, the lung cysts have frequently been confused with non‐neoplastic changes such as blebs or bullae. Herein is reported a case of multiple lung cysts associated with BHD syndrome. Detailed histopathological characteristics of the lesion are also given. The lung cysts were closely associated with the peripheral interlobular septum, visceral pleura or septal‐pleural junctional region. These cysts were partly abutting alveolar structures, and lined by a layer of alveolar epithelium. These unique microscopic features supported the notion that the BHD lung lesions are distinct from other types of bullous changes. Genomic DNA analysis indicated an aberrant sequence repeat that caused frameshift mutation. Immunohistochemistry showed the localization of folliculin, the BHD gene‐encoding protein, in macrophages and epithelial cells in the patients and normal controls lungs. Haploinsufficiency of folliculin may cause deranged alveolar development, leading to the aberrant cystic alveolar formation. The unique mutation patterns of abnormal sequence repeats in patients with BHD syndrome are also reviewed.

Disrupted Balance of Angiogenic and Antiangiogenic Signalings in Preeclampsia

The placenta plays a central role in governing local circulatory system that mediates maternal condition and fetal growth. In early gestational phases, the placenta exerts properties of invasion and neovascularization for successful placentation. Extravillous invasive trophoblasts replace uterine endometrial vasculature and establish local blood pathway to obtain oxygen and nutrients from the mother. In later phases, the placenta promotes villous angiogenesis and vascular maturation that are finely controlled by angiogenic and antiangiogenic molecules. Among various molecules involved in placental neovascularization, vascular endothelial growth factor receptors (VEGFRs) and angiotensin II receptor type 1 (AT1) mediate important signaling pathways for maternal circulatory system and fetal growth. VEGFR1 and VEGFR2 are functional receptors for placental growth factor (PlGF) and VEGF, respectively, and PlGF-VEGFR1 and VEGF-VEGFR2 interactions are disturbed in many preeclamptic patients by excess amount of soluble form of VEGFR1 (also named sFlt1), a natural PlGF/VEGF antagonist. Recent studies have disclosed that excessive sFlt1 production in the placenta and aberrant AT1 signaling in the mother are closely associated with the pathology of preeclampsia and intrauterine growth restriction (IUGR). In this paper, neovascularization of the placenta and pathological events associated with disrupted balance between angiogenic and antiangiogenic signaling in preeclampsia are discussed.