Low FOXA1 expression predicts good response to neo-adjuvant chemotherapy resulting in good outcomes for luminal HER2-negative breast cancer cases.

BACKGROUND: FOXA1 expression is a good prognostic marker for endocrine therapy in hormone-positive breast cancer. We retrospectively examined breast cancer patients with luminal human epidermal growth factor receptor 2 (HER2)-negative tumours, as defined by immunohistochemistry, who received neo-adjuvant chemotherapy (NAC) and investigated the relationship between treatment effects and FOXA1 expression. METHODS: Biopsy specimens from 103 luminal HER2-negative tumours were immunohistochemically examined. FOXA1 effects on chemo-sensitivity were also investigated employing in vitro experiments. RESULTS: FOXA1 and Ki67 expressions independently predicted a pathological complete response (pCR). Knockdown of FOXA1 by siRNA boosted the chemo-effect in oestrogen receptor-positive cells. The Cox hazards model revealed a pCR to be the strongest factor predicting a good patient outcome. CONCLUSIONS: Our present study showed low FOXA1 expression to be associated with a good response to NAC in luminal HER2-negative breast cancer. Improved outcomes of these patients suggest that NAC should be recommended to patients with low FOXA1 tumours.

Mesothelin gene expression and promoter methylation/hypomethylation in gynecological tumors.

PURPOSE: Mesothelin is a cell surface glycoprotein that is present on normal mesothelial cells and overexpressed in several cancers. In this study, we investigated the methylation/hypomethylation status in the promoter region of the mesothelin gene in gynecological tumors. METHODS: Forty-four ovarian tumor specimens and 16 cases of uterine endometrial carcinoma, and normal tissue specimens were used. Monoclonal antibody (5B2) was employed for the immunohistochemical analysis. The methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) technique was used to quantify the methylation/hypomethylation status at 20 CpG sites in the mesothelin promoter region. RESULTS: Mesothelin was expressed in 100% of serous cystadenocarcinoma and 100% of serous borderline tumor of the ovary. None of the germ cell tumors and sexcord-stromal tumors was immunoreactive. Fifty percent of endometrial carcinoma was immunoreactive for mesothelin. The average methylation of CpG sites in ovarian tumors ranged from 6-56% (median: 31%) in mesothelin-positive and 13-79% (median: 43%) in mesothelin-negative samples. In endometrial tumors, the average methylation ranged from 5-52% (median: 28%) in mesothlin-positive and from 15-67% (median: 22%) in mesothlin-negative samples. A correlation was found between mesothelin expression and the average methylation/hypomethylation status as well as methylation/hypomethylation status at four of 20 CpG sites in ovarian samples. No correlation was found in endometrial samples. CONCLUSION: We detected diverse levels of methylation/hypomethylation at CpG sites in the mesothelin promoter region in ovarian and endometrial tumors. We speculate that, although methylation/hypomethylation changes may affect its transcription, other mechanisms may synergically operate in tissue-specific expression and tumor-related mesothelin overexpression.

The G1556S-type tuberin variant suppresses tumor formation in tuberous sclerosis 2 mutant (Eker) rats despite its deficiency in mTOR inhibition.

Tuberin, a tumor-suppressor protein produced by the tuberous sclerosis gene TSC2, downregulates the Rheb-mTOR-S6K pathway (mTOR axis). Comparison of the effects of human tuberin mutations, such as G1556S, suggests that pathways other than the mTOR axis might also be involved in the pathogenesis of tuberous sclerosis. Here we test this possibility using the rat G1556S-type mutation (GSM) and a transgenic Eker (Tsc2 mutant) rat system. Cells expressing GSM-tuberin failed to downregulate the mTOR axis. GSM-tuberin had an altered localization, which underlie its reduced ability to form a complex with hamartin, and a site-specific alteration in phosphorylation status indicating diverse regulation by Akt. GSM-transgenic (GSM-Tg) rats exhibited suppression of macroscopic renal tumors following N-ethyl-N-nitrosourea treatment. Intriguingly, rats with weaker GSM-Tg expression showed microscopic cystic and pre-tumorous lesions that were restricted in size and expansion, although they had hyper-phosphorylation of ribosomal protein S6. These results highlight a novel pathway involving tuberin that regulates tumor suppression independently of the mTOR inhibitory function. Identification of such a novel pathway will provide clear implications for generation of new therapeutic targets in the treatment of these tumors.

Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein.

Birt-Hogg-Dubé (BHD) syndrome is characterized by the development of pneumothorax, hair folliculomas and renal tumors and the responsible BHD gene is thought to be a tumor suppressor. The function of folliculin (Flcn), encoded by BHD, is totally unknown, although its interaction with Fnip1 has been reported. In this study, we identified a novel protein binding to Flcn, which is highly homologous to Fnip1, and which we named FnipL (recently reported in an independent study as Fnip2). The interaction between FnipL/Fnip2 and Flcn may be mediated mainly by the C-terminal domains of each protein as is the case for the Flcn-Fnip1 interaction. FnipL/Fnip2 and Flcn were located together in the cytoplasm in a reticular pattern, although solely expressed Flcn was found mainly in the nucleus. Cytoplasmic retention of Flcn was canceled with C-terminal truncation of FnipL/Fnip2, suggesting that FnipL/Fnip2 regulates Flcn distribution through their complex formation. By the employment of siRNA, we observed a decrease in S6K1 phosphorylation in the BHD-suppressed cell. We also observed a decrease in S6K1 phosphorylation in FNIP1- and, to a lesser extent, in FNIPL/FNIP2-suppressed cells. These results suggest that Flcn-FnipL/Fnip2 and Flcn-Fnip1 complexes positively regulate S6K1 phosphorylation and that FnipL/Fnip2 provides an important clue to elucidating the function of Flcn and the pathogenesis of BHD.

Birt-Hogg-Dubé gene mutations in human endometrial carcinomas with microsatellite instability.

Birt-Hogg-Dubé (BHD) syndrome is a rare form of autosomal dominantly inherited genodermatosis characterized by benign hamartomatous skin lesions named fibrofolliculomas, and an increased risk for developing pulmonary cyst/pneumothorax and various forms of renal cell carcinoma. Many of the patients harbour insertion/deletion mutations in the hypermutable poly(C)8 tract in exon 11 of the BHD gene. This mutational hot spot is also reported to be a target of mutation in microsatellite instability (MSI) sporadic colorectal cancer. To test the hypothesis that the BHD gene is also a mutational target in sporadic endometrial carcinoma with microsatellite instability, 139 cases of sporadic endometrial carcinoma were screened for MSI status, and mutations of the poly(C)8 tract in exon 11 as well as other coding exons of the BHD gene. The poly(G)8 tract of the BAX gene, the poly(C)8 tract of MSH6, and methylation status of hMLH1 were also assessed. Thirty-nine of 139 cases (28%) showed MSI. Mutations in the poly(C)8 tract of BHD were detected in five of the 39 MSI cases (12.8%). Of these, one showed additional mutation in exon 4, possibly satisfying the two-hit hypothesis of tumour suppressor genes. BAX gene mutation was detected in ten of the 39 MSI cases (25.6%). Four tumours showed both BAX and BHD mutations, and a significant positive association was found between mutations of the two genes. No association was found between BHD status and MSH6 mutation or hMLH1 methylation. When multiple foci were microdissected and individually screened for mutation, BHD mutations were shown to have been acquired during tumour progression, after mutation of the BAX gene, in three of five cases. Taken together, these findings show that the BHD gene is a target gene in MSI endometrial carcinoma. However, its mutational frequency is lower than that of BAX, and BHD mutation tends to occur during neoplastic progression after the acquisition of mutations in another MSI target gene, BAX.

Transgenic rescue from embryonic lethality and renal carcinogenesis in the Nihon rat model by introduction of a wild-type Bhd gene.

We recently reported that a germline insertion of a single nucleotide in the rat homologue of the human Birt-Hogg-Dubé gene (BHD) gives rise to dominantly inherited cancer in the Nihon rat model. In this study, we constructed transgenic Nihon rats with introduction of a wild-type Bhd gene to ascertain whether suppression of the Nihon phenotype is possible. Rescue from embryonic lethality of mutant homozygotes (Nihon/Nihon) and suppression of renal carcinogenesis in heterozygotes (Nihon/+) were both observed, defining the germline Bhd mutation in the Nihon rat as an embryonal lethal and tumor predisposing mutation. This transgenic rescue system will be useful to analyse Bhd gene function, its relation to tumorigenesis in vivo, and genetic-environmental interactions in carcinogenesis.

Allelic loss analysis of early-stage flat-type colorectal tumors.

BACKGROUND: Flat-type colorectal tumors are rare, but are known for their unusual flat morphology and aggressive clinical behavior despite their small size. To identify distinct genetic alterations, loss of heterozygosity (LOH) analysis was performed on microdissected tissues. MATERIALS AND METHODS: DNA was extracted from multiple microdissected foci in 43 cases of early-stage flat-type colorectal tumors and LOH analysis was performed on 2q, 4q, 5q, 12q, 14q, 15q, 17p, 18q, 18p and 22q. RESULTS: LOH patterns were detected in one of two forms: (i) homogeneous LOH throughout the microdissected foci, which indicated the early acquisition of LOH; and (ii) heterogeneous LOH, which were detected in a part of analyzed foci. Homogeneous and heterogeneous LOH were most frequently detected on 17p (92%) followed by 18q (81%), 18p (81%), 5q (61%), 22q (51%), 14q (44%), 15q (41%), 2q (39%), 12q (36%) and 4q (32%). Homogeneous LOH was detected most frequently on 17p (68%) followed by 18p (53%), 18q (53%), 22q (34%) and 12q (27%). The average fractional allelic loss (FAL) for heterogeneous and homogeneous LOH was 0.57 and the average FAL for homogeneous LOH was 0.37. CONCLUSIONS: Early flat-type colorectal tumors frequently shows the early occurrence of multiple LOH including 17p, 18p, 18q and 22q, which is coupled with additional LOH of other loci either simultaneously or in the early clonal progression phase. The extent and sequences of LOH may be the mechanisms responsible for the aggressive clinical behaviors of these tumors.

A patient with TSC1 germline mutation whose clinical phenotype was limited to lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis (LAM) can occur as in isolated form (sporadic LAM) or as a pulmonary manifestation of tuberous sclerosis complex (TSC) (TSC-associated LAM). Recent studies, however, revealed that both forms of LAM are genetically related but that sporadic LAM is a distinct clinical entity caused by somatic mutations of TSC2 (not TSC1) rather than a forme fruste of TSC carrying either of the TSC1 or TSC2 germline mutations. METHOD: Case presentation and in-depth molecular and histopathological examinations. A 34-year-old Japanese woman was diagnosed as having pulmonary lymphangioleiomyomatosis (LAM) when bilateral pneumothoraces were surgically treated in 1992. Although slowly progressive renal disfunction was observed due to bilateral multiple renal cysts during the past 4 years, she had no other clinical features of TSC and was diagnosed as having sporadic LAM with multiple renal cysts of undetermined aetiology. Her subsequent clinical course was complicated by an endobrochial carcinoid tumour, which eventually resulted in her death in June 1999 due to massive haemoptysis. RESULTS: Postmortem examination revealed the presence of LAM lesions in the lungs, mediastinal lymph nodes, kidneys and uterus. Diffuse renal LAM lesions are presumed to generate multiple renal cysts by constricting the nephron rather than epithelial hyperplasia obstructing lumina, which is analysis of the TSC genes demonstrated that she did not have TSC2/PKD1 contiguous gene syndrome but had a TSC1 germline mutation (Sato T et al. J Hum Genet 2002; 47: 20-8) that had occured de novo. CONCLUSION: This patient therefore illustrates that clinical manifestations of TSC are sufficiently diverse as to allow a forme fruste of TSC that mimics sporadic LAM and that TSC1 mutation can cause multiple renal cysts resulting in renal failure.

A novel renal carcinoma predisposing gene of the Nihon rat maps on chromosome 10.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley (SD) strain in Japan, and named the "Nihon" rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross animals. Our present data clearly show that the Nihon gene is genetically linked to interleukin-3 (IL3) gene (chi(2) = 93.6, Lod score = 25.16), lethal (2) giant larvae (LLGL1) locus (chi(2) = 109.0, Lod score = 31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (chi(2) = 90.6, Lod score = 23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)--21.3 cM--IL3 gene (human 5q23-31)--4.4 cM--Nihon gene--0.9 cM--LLGL1 locus (human 17p11.2)--4.4 cM--MYHSE gene (human 17p13.1). We also detected loss of the wild-type allele at the MYHSE locus, fitting Knudson's "two hit" model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

Recombination hot spot of hepatitis B virus genome binds to members of the HMG domain protein family and the Y box binding protein family; implication of these proteins in genomic instability.

OBJECTIVE: Previously we hypothesized that the occurrence of hepatocellular carcinoma (HCC) is enhanced by genomic instability induced by the integrated hepatitis B virus (HBV) DNA. Using an in vitro recombination assay, we showed that a subgenomic fragment of HBV DNA designated 15AB (nt1855-1914) is indispensable for in vitro recombination, and also showed the existence of 15AB binding protein. On the assumption that the 15AB binding protein may be a candidate cellular recombinogenic protein which accelerates genomic instability and hepatocarcinogenesis, we tried to isolate it by southwestern screening. RESULTS AND CONCLUSION: We obtained several positive clones including mouse upstream binding factor (UBF) and DNA binding protein A (dbpA). UBF belongs to an HMG domain protein family and dbpA belongs to a Y box binding protein family. 15AB binding seemed to be mediated by the conserved DNA binding domains in these families, because other members in the families such as HMG1 and YB-1 also bound to 15AB. We report them here because several documents have already suggested the possible association of these families and DNA recombination.

Pathogenesis of multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis and association with tuberous sclerosis genes TSC1 and TSC2.

Tuberous sclerosis (TSC) is a rare, genetically determined disorder / familial tumor syndrome, currently diagnosed using specific clinical criteria proposed by Gomez, including the presence of multiorgan hamartomas. Pulmonary involvement in TSC is well known as pulmonary lymphangioleiomyomatosis (LAM), which has an incidence of 1-2.3% in TSC patients. LAM has immunohistochemical expression of both smooth-muscle actin and a monoclonal antibody specific for human melanoma, HMB-45. It has recently been reported that multifocal micronodular pneumocyte hyperplasia (MMPH) associated with TSC should be considered as a distinct type of lung lesion, whether it occurs with or without LAM. Two predisposing genes have been found in families affected by TSC; approximately half of the families show linkage to TSC1 at 9q34.3, and the other half show linkage to TSC2 at 16p13.3. TSC genes are considered to be tumor suppressor genes, and mutations in them may lead to abnormal differentiation and proliferation of cells. Tuberin, the TSC2 gene product, has recently been found to be expressed in LAM and MMPH. In this article we discuss the histogenesis and genetic abnormalities of neoplastic lesions associated with TSC, and we review the current understanding of the pathogenesis of pulmonary hamartomatous lesions such as LAM and MMPH in TSC.

Dendritic cells and chronic hepatitis virus carriers.

Many individuals infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) are unable to clear these viruses following an acute infection and become chronically infected. There are more than 400 million HBV and HCV carriers in the world and a considerable number of these patients would eventually develop more severe complications like liver cirrhosis and hepatocellular carcinoma. It is not clearly known how an individual develops a chronic hepatitis virus carrier state; however, a defective immune response of the host is thought to play a critical role in the underlying pathogenetic mechanism. On the other hand, dendritic cells (DCs), the most potent antigen-presenting cells, are widely distributed in both lymphoid and nonlymphoid tissues. Recognition of the microbes or microbial antigens by DCs is one of critical events for the initiation of an immune response. DCs also play a cardinal role during the progression and termination of an immune response. The aim of this overview is to provide information regarding the role of DCs in the pathogenesis of chronic hepatitis due to HBV and HCV in humans and in animal models of HBV and HCV carrier states. First, we summarize our current understanding of the pathogenesis of hepatitis virus carrier states and also of general properties of DCs. Next, we discuss the data on the phenotypes and functions of DCs in both human and murine HBV and HCV carriers. We also discuss vaccine therapy in murine HBV carriers because activation of DCs due to vaccination-initiated HBsAg-specific immune responses in HBV transgenic mice (HBV-Tg), which in turn resulted in complete clearance of hepatitis B surface antigen and hepatitis B e antigen and decreased levels of HBV DNA in some HBV-Tg. Finally, we discuss the extracted questions and future research directions.

A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice.

Tuberous sclerosis (TS) is characterized by the development of hamartomas in various organs and is caused by a germ-line mutation in either TSC1 or TSC2 tumor suppressor genes. From the symptomatic resemblance among TS patients, involvement of TSC1 and TSC2 products in a common pathway has been suggested. Here, to analyze the function of the Tsc1 product, we established a line of Tsc1 (TSC1 homologue) knockout mouse by gene targeting. Heterozygous Tsc1 mutant (Tsc1(+/-)) mice developed renal and extra-renal tumors such as hepatic hemangiomas. In these tumors, loss of wild-type Tsc1 allele was observed. Homozygous Tsc1 mutants died around embryonic days 10.5-11.5, frequently associated with neural tube unclosure. As a whole, phenotypes of Tsc1 knockout mice resembled those of Tsc2 knockout mice previously reported, suggesting that the presumptive common pathway for Tsc1 and Tsc2 products may also exist in mice. Notably, however, development of renal tumors in Tsc1(+/-) mice was apparently slower than that in Tsc2(+/-) mice. The Tsc1 knockout mouse described here will be a useful model to elucidate the function of Tsc1 and Tsc2 products as well as pathogenesis of TS.

Multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis with a TSC2 gene.

A 45-year-old woman with a long-standing diagnosis of tuberous sclerosis (TSC) is presented. She has multifocal micronodular pneumocyte hyperplasia (MMPH) and lymphangioleiomyomatosis (LAM) of the lung, together with the detection of TSC2 gene mutation. During surgery for spontaneous pneumothorax, an open-lung biopsy was performed. Micronodules were well defined, measuring approximately 4 mm in diameter. These MMPHs were histologically composed of papillary proliferation of Type II pneumocytes, with positive immunoreactivity of keratin and surfactant apoprotein. The cystlike spaces, with dilatation and destruction of air spaces, were diffusely formed, and the walls were composed of the spindle cells. Such LAM showed positive immunoreactivity for HMB-45 (a monoclonal antibody specific for human melanoma) and tuberin (the gene product of TSC2). On germline mutation analysis using leukocytes of the present patient, a TSC2 gene mutation was confirmed as a deletion of G (or g) on Exon 9 by polymerase chain reaction-single-strand conformational polymorphism. However, no mutation was detected in her son. With microdissection analysis using paraffin-embedding lung tissues, LOH of the TSC2 gene preliminarily was detected in a LAM lesion but not in MMPH. It is suggested that MMPH, in addition to LAM, could be another pulmonary lesion in TSC patients and that the detection of TSC2 and/or TSC1 gene could essentially be useful for the pathogenesis of MMPH and LAM in TSC patients.

Multistep renal carcinogenesis as gene expression disease in tumor suppressor TSC2 gene mutant model--genotype, phenotype and environment.

Cancer is an inheritable disorder of somatic cells. Environment and heredity both operate in the origins of human cancer. These environmental and genetic determinants of cancer can be classified into four groups designated "Oncodemes" [1]. Oncodeme 1 is the irreducible "background" level of cancer due to spontaneous mutagenesis. Oncodeme 2 is "environmentally induced" cancer, whose causative agents are chemical carcinogens, radiation and viruses. Oncodeme 3 is basically "environmentally induced" cancer, but there are genetically determined differences among persons, e.g. the activation or inactivation of carcinogenes. Most human cancers are believed to belong to Oncodemes 2 and/or 3 (about 80%), for which the probability of the occurrence of the initial carcinogenic step(s) is increased, although the number of steps is not decreased. Oncodeme 1 would contain the approximately 20% that would remain if "environmentally induced" cancers (Oncodeme 2 and/or 3) were prevented. Lastly, Oncodeme 4 is "hereditary" cancer. Hereditary cancers could prove valuable in elucidating carcinogenesis, even though only a small proportion of cancers belong to this group. Here, we present a unique animal model of Oncodeme 4 for the study of problems in carcinogenesis; e.g. cell stage and tissue/cell-type-specific tumorigenesis, multistep carcinogenesis, species-specific differences in tumorigenesis, modifier gene(s) in renal carcinogenesis and cancer prevention.

Suppression of cytochrome P450 1A1 and 4A1 gene expression in renal carcinomas of TSC2 gene mutant (Eker) rats.

In the kidney, cytochrome P450 (CYP) is involved in arachidonic acid metabolism and the maintenance of homeostasis, but only scarce information is available as to how CYP expression is altered in rodent renal carcinomas (RCs). TSC2 gene mutant (Eker) rat RCs are an example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. In the present study, the expression of CYP in Eker RCs was studied. In the normal kidney, CYP 1A1 and 4A1 mRNAs were expressed, but this expression was suppressed in spontaneously-induced Eker RCs and in cell line Lk9dL and Lk9dR. In Lk9dL and Lk9dR, Ah receptor nuclear translocator and haemoxygenase-1 mRNAs were expressed, but this expression was inconsistent in spontaneously-induced Eker RCs. The present results showed the suppression of CYP 1A1 and 4A1 mRNA expression in spontaneously-induced Eker RCs and this suppression indicates altered metabolic conditions in Eker RCs.

Hepatoprotective drugs for the treatment of virus-induced chronic hepatitis: from hypercarcinogenic state to hypocarcinogenic state.

Interferon (IFN)-based therapy is a standard treatment for chronic hepatitis caused by hepatitis C virus (HCV) infection. This treatment is effective in approximately 30-40% of the patients and using ribavirin in combination with IFN increases the rate of sustained virologic clearance. For the remaining patients, glycyrrhizin is often used. Glycyrrhizin is known to prevent the development of hepatocellular carcinoma (HCC), but glycyrrhizin is usually administered intravenously. Drugs that are effective by oral administration are convenient for patients for long-term administration, and development of more effective drugs than glycyrrhizin is preferable. However, studies on drugs for the treatment of hepatitis are not actively conducted, and promotion of the study of drugs in this area is encouraging. For that reason, we show our approach to study drugs for the treatment of hepatitis. We analyzed the effect of glycyrrhizin on hepatitis as a standard chemical using the mouse liver injury model. Based on this, we screened drugs and found that a coumarin derivative seems to be one of model chemicals for the treatment of hepatitis.