Exploring the roles of MACIT and multiplexin collagens in stem cells and cancer.

The extracellular matrix (ECM) is ubiquitously involved in neoplastic transformation, tumour growth and metastatic dissemination, and the interplay between tumour and stromal cells and the ECM is now considered crucial for the formation of a tumour-supporting microenvironment. The 28 different collagens (Col) form a major ECM protein family and display extraordinary functional diversity in tissue homeostasis as well as in pathological conditions, with functions ranging from structural support for tissues to regulatory binding activities and storage of biologically active cryptic domains releasable through ECM proteolysis. Two subfamilies of collagens, namely the plasma membrane-associated collagens with interrupted triple-helices (MACITs, including ColXIII, ColXXIII and ColXXV) and the basement membrane-associated collagens with multiple triple-helix domains with interruptions (multiplexins, including ColXV and ColXVIII), have highly interesting regulatory functions in tissue and organ development, as well as in various diseases, including cancer. An increasing, albeit yet sparse, data suggest that these collagens play crucial roles in conveying regulatory signals from the extracellular space to cells. We summarize here the current knowledge about MACITs and multiplexins as regulators of stemness and oncogenic processes, as well as their roles in influencing cell fate decisions in healthy and cancerous tissues. In addition, we present a bioinformatic analysis of the impacts of MACITs and multiplexins transcript levels on the prognosis of patients representing a wide array of malignant diseases, to aid future diagnostic and therapeutic efforts.

Deletion of Col15a1 Modulates the Tumour Extracellular Matrix and Leads to Increased Tumour Growth in the MMTV-PyMT Mouse Mammary Carcinoma Model.

Basement membrane (BM) zone-associated collagen XV (ColXV) has been shown to suppress the malignancy of tumour cells, and its restin domain can inhibit angiogenesis. In human breast cancer, as well as in many other human carcinomas, ColXV is lost from the epithelial BM zone prior to tumour invasion. Here, we addressed the roles of ColXV in breast carcinogenesis using the transgenic MMTV-PyMT mouse mammary carcinoma model. We show here for the first time that the inactivation of Col15a1 in mice leads to changes in the fibrillar tumour matrix and to increased mammary tumour growth. ColXV is expressed by myoepithelial and endothelial cells in mammary tumours and is lost from the ductal BM along with the loss of the myoepithelial layer during cancer progression while persisting in blood vessels and capillaries, even in invasive tumours. However, despite the absence of anti-angiogenic restin domain, neovascularisation was reduced rather than increased in the ColXV-deficient mammary tumours compared to controls. We also show that, in robust tumour cell transplantation models or in a chemical-induced fibrosarcoma model, the inactivation of Col15a1 does not affect tumour growth or angiogenesis. In conclusion, our results support the proposed tumour suppressor function of ColXV in mammary carcinogenesis and reveal diverse roles of this collagen in different cancer types.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

KEY POINTS: Extracellular matrix is highly remodelled in obesity and associates with the development of metabolic disorders, such as insulin resistance. Previously, we have shown that the lack of specific collagen XVIII isoforms impairs adipocyte differentiation in mice. Here, we show that mice lacking the medium and long isoforms of collagen XVIII develop insulin resistance and glucose intolerance and show elevated serum triglycerides and fat accumulation in the liver. We report that collagen XVIII-deficient mice have increased heat production at low temperatures. These results reveal a new role for collagen XVIII in the regulation of glucose and lipid metabolism, and they expand the understanding of the development of metabolic disorders. ABSTRACT: Liver and adipose tissues play important roles in the regulation of systemic glucose and lipid metabolism. Extracellular matrix synthesis and remodelling are significantly altered in these tissues in obesity and type 2 diabetes. Collagen XVIII is a ubiquitous extracellular matrix component, and it occurs in three isoforms which differ in terms of molecular size, domain structure and tissue distribution. We recently showed that, in mice, the lack of collagen XVIII, and especially its medium and long isoforms, leads to reduced adiposity and dyslipidaemia. To address the metabolic consequences of these intriguing observations, we assessed whole-body glucose homeostasis in mice challenged with a high-fat diet and in normal physiological conditions. We observed that, in the high caloric diet, the overall adiposity was decreased by 30%, serum triglyceride values were threefold higher and the steatotic area in liver was twofold larger in collagen XVIII knockout mice compared with controls. We demonstrated that mice lacking either all three collagen XVIII isoforms, or specifically, the medium and long isoforms develop insulin resistance and glucose intolerance. Furthermore, we found that ablation of collagen XVIII leads to increased heat production in low temperatures and to reduction of the high blood triglyceride levels of the knockout mice to the level of wild-type mice. Our data indicate that collagen XVIII plays a role in the regulation of glucose tolerance, insulin sensitivity and lipid homeostasis, principally through its ability to regulate the expansion of the adipose tissue. These findings advance the understanding of metabolic disorders.

Collagens XV and XVIII show different expression and localisation in cutaneous squamous cell carcinoma: type XV appears in tumor stroma, while XVIII becomes upregulated in tumor cells and lost from microvessels.

As the second most common skin malignancy, cutaneous squamous cell carcinoma (cSCC) is an increasing health concern, while its pathogenesis at molecular level remains largely unknown. We studied the expression and localisation of two homologous basement membrane (BM) collagens, types XV and XVIII, at different stages of cSCC. These collagens are involved in angiogenesis and tumorigenesis, but their role in cancer development is incompletely understood. Quantitative RT-PCR analysis revealed upregulation of collagen XVIII, but not collagen XV, in primary cSCC cells in comparison with normal human epidermal keratinocytes. In addition, the Ha-ras-transformed invasive cell line II-4 expressed high levels of collagen XVIII mRNA, indicating upregulation in the course of malignant transformation. Immunohistochemical analyses of a large human tissue microarray material showed that collagen XVIII is expressed by tumor cells from grade 1 onwards, while keratinocytes in normal skin and in premalignant lesions showed negative staining for it. Collagen XV appeared instead as deposits in the tumor stroma. Our findings in human cSCCs and in mouse cSCCs from the DMBA-TPA skin carcinogenesis model showed that collagen XVIII, but not collagen XV or the BM markers collagen IV or laminin, was selectively reduced in the tumor vasculature, and this decrease associated significantly with cancer progression. Our results demonstrate that collagens XV and XVIII are expressed in different sites of cSCC and may contribute in a distinct manner to processes related to cSCC tumorigenesis, identifying these collagens as potential biomarkers in the disease.

A recurrent mutation in PALB2 in Finnish cancer families.

BRCA1, BRCA2 and other known susceptibility genes account for less than half of the detectable hereditary predisposition to breast cancer. Other relevant genes therefore remain to be discovered. Recently a new BRCA2-binding protein, PALB2, was identified. The BRCA2-PALB2 interaction is crucial for certain key BRCA2 DNA damage response functions as well as its tumour suppression activity. Here we show, by screening for PALB2 mutations in Finland that a frameshift mutation, c.1592delT, is present at significantly elevated frequency in familial breast cancer cases compared with ancestry-matched population controls. The truncated PALB2 protein caused by this mutation retained little BRCA2-binding capacity and was deficient in homologous recombination and crosslink repair. Further screening of c.1592delT in unselected breast cancer individuals revealed a roughly fourfold enrichment of this mutation in patients compared with controls. Most of the mutation-positive unselected cases had a familial pattern of disease development. In addition, one multigenerational prostate cancer family that segregated the c.1592delT truncation allele was observed. These results indicate that PALB2 is a breast cancer susceptibility gene that, in a suitably mutant form, may also contribute to familial prostate cancer development.

Targeting collagen XVIII improves the efficiency of ErbB inhibitors in breast cancer models.

The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII was markedly upregulated in human breast cancer (BC) and was closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of epidermal growth factor receptor tyrosine kinase (ErbB) signaling and show that it forms a complex with ErbB1 and -2 (also known as EGFR and human epidermal growth factor receptor 2 [HER2]) and α6-integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies using Col18a1 mouse models crossed with the mouse mammary tumor virus-polyoma virus middle T antigen (MMTV-PyMT) mammary carcinogenesis model showed that ColXVIII promoted BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.

Upregulated integrin α11 in the stroma of cutaneous squamous cell carcinoma promotes skin carcinogenesis.

Integrin α11ß1 is a collagen-binding integrin that is needed to induce and maintain the myofibroblast phenotype in fibrotic tissues and during wound healing. The expression of the α11 is upregulated in cancer-associated fibroblasts (CAFs) in various human neoplasms. We investigated α11 expression in human cutaneous squamous cell carcinoma (cSCC) and in benign and premalignant human skin lesions and monitored its effects on cSCC development by subjecting α11-knockout (Itga11-/- ) mice to the DMBA/TPA skin carcinogenesis protocol. α11-deficient mice showed significantly decreased tumor cell proliferation, leading to delayed tumor development and reduced tumor burden. Integrin α11 expression was significantly upregulated in the desmoplastic tumor stroma of human and mouse cSCCs, and the highest α11 expression was detected in high-grade tumors. Our results point to a reduced ability of α11-deficient stromal cells to differentiate into matrix-producing and tumor-promoting CAFs and suggest that this is one causative mechanism underlying the observed decreased tumor growth. An unexpected finding in our study was that, despite reduced CAF activation, the α11-deficient skin tumors were characterized by the presence of thick and regularly aligned collagen bundles. This finding was attributed to a higher expression of TGFß1 and collagen crosslinking lysyl oxidases in the Itga11-/- tumor stroma. In summary, our data suggest that α11ß1 operates in a complex interactive tumor environment to regulate ECM synthesis and collagen organization and thus foster cSCC growth. Further studies with advanced experimental models are still needed to define the exact roles and molecular mechanisms of stromal α11ß1 in skin tumorigenesis.

Collagen XV, a multifaceted multiplexin present across tissues and species.

Type XV collagen is a non-fibrillar collagen that is associated with basement membranes and belongs to the multiplexin subset of the collagen superfamily. Collagen XV was initially studied because of its sequence homology with collagen XVIII/endostatin whose anti-angiogenic and anti-tumorigenic properties were subjects of wide interest in the past years. But during the last fifteen years, collagen XV has gained growing attention with increasing number of studies that have attributed new functions to this widely distributed collagen/proteoglycan hybrid molecule. Despite the cumulative evidence of its functional pleiotropy and its evolutionary conserved function, no review compiling the current state of the art about collagen XV is currently available. Here, we thus provide the first comprehensive view of the knowledge gathered so far on the molecular structure, tissue distribution and functions of collagen XV in development, tissue homeostasis and disease with an evolutionary perspective. We hope that our review will open new roads for promising research on collagen XV in the coming years.

Toward understanding scarless skin wound healing and pathological scarring.

The efficient healing of skin wounds is crucial for securing the vital barrier function of the skin, but pathological wound healing and scar formation are major medical problems causing both physiological and psychological challenges for patients. A number of tightly coordinated regenerative responses, including haemostasis, the migration of various cell types into the wound, inflammation, angiogenesis, and the formation of the extracellular matrix, are involved in the healing process. In this article, we summarise the central mechanisms and processes in excessive scarring and acute wound healing, which can lead to the formation of keloids or hypertrophic scars, the two types of fibrotic scars caused by burns or other traumas resulting in significant functional or aesthetic disadvantages. In addition, we discuss recent developments related to the functions of activated fibroblasts, the extracellular matrix and mechanical forces in the wound environment as well as the mechanisms of scarless wound healing. Understanding the different mechanisms of wound healing is pivotal for developing new therapies to prevent the fibrotic scarring of large skin wounds.

Germline alterations in the CLSPN gene in breast cancer families.

About 5-10% of breast cancer is thought to be due to an inherited disease predisposition. Currently known genes account for less than half of the hereditary cases. Claspin, a tumor suppressor protein encoded by the CLSPN gene, is involved in monitoring of replication and sensoring of DNA damage and cooperates with CHK1 and BRCA1. Association with certain cell proliferation stimulatory features has also been described. Many previously identified susceptibility factors act in similar functional pathways as claspin, suggesting possible involvement of CLSPN in heritable breast cancer susceptibility. Here we have screened affected index cases from 125 Finnish cancer families for germline defects in CLSPN using conformation sensitive gel electrophoresis (CSGE) and direct sequencing. Altogether seven different sequence changes were observed, but none of them appeared to associate with breast cancer susceptibility. To our knowledge, this is the first study reporting the mutation screening of the CLSPN gene in familial breast cancer cases.

Germline alterations in the 53BP1 gene in breast and ovarian cancer families.

The conserved TP53-binding protein 1 (53BP1) is a central mediator of the DNA damage checkpoint and appears to be one of the sensors of DNA double-strand breaks (DSBs). Improper processing of DSBs can result in loss or rearrangement of genetic information, leading to cell death or tumorigenesis. 53BP1 interacts with both TP53 and ATM, key proteins involved in the monitoring of genomic integrity and regulation of apoptosis. 53BP1 is also required for the formation of BRCA1 foci and the C-terminal part of these two proteins display significant homology. Based on its biological function, the 53BP1 gene is a good candidate for being involved in cancer susceptibility. Consequently, in the current study patients belonging to 126 breast and/or ovarian cancer families were screened for germline mutations in the entire coding region of the 53BP1 gene. A number of sequence variants were found, but none of them appeared to associate with cancer predisposition. To our knowledge this is the first comprehensive screening of 53BP1 mutations in familial breast and ovarian cancer cases.

GATA4-dependent organ-specific endothelial differentiation controls liver development and embryonic hematopoiesis.

Microvascular endothelial cells (ECs) are increasingly recognized as organ-specific gatekeepers of their microenvironment. Microvascular ECs instruct neighboring cells in their organ-specific vascular niches through angiocrine factors, which include secreted growth factors (angiokines), extracellular matrix molecules, and transmembrane proteins. However, the molecular regulators that drive organ-specific microvascular transcriptional programs and thereby regulate angiodiversity are largely elusive. In contrast to other ECs, which form a continuous cell layer, liver sinusoidal ECs (LSECs) constitute discontinuous, permeable microvessels. Here, we have shown that the transcription factor GATA4 controls murine LSEC specification and function. LSEC-restricted deletion of Gata4 caused transformation of discontinuous liver sinusoids into continuous capillaries. Capillarization was characterized by ectopic basement membrane deposition, formation of a continuous EC layer, and increased expression of VE-cadherin. Correspondingly, ectopic expression of GATA4 in cultured continuous ECs mediated the downregulation of continuous EC-associated transcripts and upregulation of LSEC-associated genes. The switch from discontinuous LSECs to continuous ECs during embryogenesis caused liver hypoplasia, fibrosis, and impaired colonization by hematopoietic progenitor cells, resulting in anemia and embryonic lethality. Thus, GATA4 acts as master regulator of hepatic microvascular specification and acquisition of organ-specific vascular competence, which are indispensable for liver development. The data also establish an essential role of the hepatic microvasculature in embryonic hematopoiesis.

Identification of a common polymorphism in the TopBP1 gene associated with hereditary susceptibility to breast and ovarian cancer.

Besides BRCA1 and BRCA2 other genes are also likely to be involved in hereditary predisposition to breast and/or ovarian cancer. TopBP1 (topoisomerase IIbeta binding protein 1) displays sequence homology as well as functional similarities with BRCA1, and the two proteins have been suggested to function partly in the same cellular processes. TopBP1 is crucial for DNA damage and replication checkpoint controls. Based on its biological significance, we reasoned that TopBP1 is a plausible susceptibility gene for hereditary breast and/or ovarian cancer and therefore screened affected index cases from 125 Finnish cancer families for germline changes by conformation sensitive gel electrophoresis (CSGE). Altogether 19 different sequence alterations were detected. A novel heterozygous Arg309Cys variant was observed at elevated frequency in the familial cancer cases compared to healthy controls (15.2% versus 7.0%; P=0.002). Current results suggest that Arg309Cys is a commonly occurring germline alteration possibly associated with a slightly increased breast and/or ovarian cancer risk. This is the first study reporting mutation screening of the TopBP1 gene in a familial cancer material.

Screening for RAD51 and BRCA2 BRC repeat mutations in breast and ovarian cancer families.

Together, germline mutations in the two major susceptibility genes BRCA1 and BRCA2 account for approximately 20-30% and 70-80% of the familial breast and ovarian cancer cases, respectively. This indicates involvement of additional susceptibility genes, perhaps in combination with a polygenic effect. However, it is also possible that part of the mutations disrupting BRCA1 and BRCA2 function still remains to be discovered. In response to double-strand DNA damage the co-operation between RAD51 and BRCA2 is of great importance, and the conserved BRC repeat motifs in BRCA2 are crucial for this interaction. In the current study, patients belonging to 126 breast and/or ovarian cancer families were screened for RAD51 and BRCA2 BRC repeat mutations in order to uncover aberrations that may contribute to hereditary cancer susceptibility. The performed study revealed several novel alterations, however, none of them appeared to be disease-related. Thus, it seems likely that germline mutations in the highly conserved RAD51 gene are extremely rare and generally poorly tolerated.

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging: A Proof-of-Concept Study.

BACKGROUND AND AIMS: Early detection of fibrosis is important in identifying individuals at risk for advanced liver disease in non-alcoholic fatty liver disease (NAFLD). We tested whether second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy, detecting fibrillar collagen and fat in a label-free manner, might allow automated and sensitive quantification of early fibrosis in NAFLD. METHODS: We analyzed 32 surgical biopsies from patients covering histological fibrosis stages 0-4, using multimodal label-free microscopy. Native samples were visualized by SHG and CARS imaging for detecting fibrillar collagen and fat. Furthermore, we developed a method for quantitative assessment of early fibrosis using automated analysis of SHG signals. RESULTS: We found that the SHG mean signal intensity correlated well with fibrosis stage and the mean CARS signal intensity with liver fat. Little overlap in SHG signal intensities between fibrosis stages 0 and 1 was observed. A specific fibrillar SHG signal was detected in the liver parenchyma outside portal areas in all samples histologically classified as having no fibrosis. This signal correlated with immunohistochemical location of fibrillar collagens I and III. CONCLUSIONS: This study demonstrates that label-free SHG imaging detects fibrillar collagen deposition in NAFLD more sensitively than routine histological staging and enables observer-independent quantification of early fibrosis in NAFLD with continuous grading.

Mutation analysis of the ATR gene in breast and ovarian cancer families.

INTRODUCTION: Mutations in BRCA1, BRCA2, ATM, TP53, CHK2 and PTEN account for only 20-30% of the familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. The ATR (ataxia-telangiectasia- and Rad3-related) kinase is essential for the maintenance of genomic integrity. It functions both in parallel and cooperatively with ATM, but whereas ATM is primarily activated by DNA double-strand breaks induced by ionizing radiation, ATR has been shown to respond to a much broader range of DNA damage. Upon activation, ATR phosphorylates several important tumor suppressors, including p53, BRCA1 and CHK1. Based on its central function in the DNA damage response, ATR is a plausible candidate gene for susceptibility to cancer. METHODS: We screened the entire coding region of the ATR gene for mutations in affected index cases from 126 Finnish families with breast and/or ovarian cancer, 75 of which were classified as high-risk and 51 as moderate-risk families, by using conformation sensitive gel electrophoresis and direct sequencing. RESULTS: A large number of novel sequence variants were identified, four of which -- Glu254Gly, Ser1142Gly, IVS24-48G>A and IVS26+15C>T -- were absent from the tested control individuals (n = 300). However, the segregation of these mutations with the cancer phenotype could not be confirmed, partly because of the lack of suitable DNA samples. CONCLUSION: The present study does not support a major role for ATR mutations in hereditary susceptibility to breast and ovarian cancer.

Evaluation of the role of Finnish ataxia-telangiectasia mutations in hereditary predisposition to breast cancer.

Biallelic mutations in the ataxia-telangiectasia mutated (ATM) gene result in ataxia-telangiectasia (A-T). Studies on A-T families have shown that obligate female carriers have increased risk of developing breast cancer. Here we have evaluated the role of known Finnish ATM germ line mutations as possible breast cancer predisposing alleles outside A-T families by analyzing their prevalence in large cohorts of familial and unselected breast cancer cases. Of seven different alterations, two were observed in the studied breast cancer material. ATM 6903insA (causing protein truncation) was seen in 3/541 familial and 5/1124 unselected cases, but not among healthy population controls (0/1107). 7570G>C (Ala2524Pro) occurred in 1/541 familial and 2/1124 unselected cases compared with 1/1107 in controls. Additionally, 8734A>G (Arg2912Gly) associated previously with breast cancer susceptibility and suggested to be causative also for A-T was detected in 2/541 of familial cases, but not in unselected cases (0/1124) or controls (0/1107). In total, heterozygous ATM mutation carriers were observed in 6/541 familial [P = 0.006, odds ratio (OR) 12.4, 95% confidence interval (CI) 1.5-103.3) and 7/1124 unselected cases (P = 0.07, OR 6.9, 95% CI 0.9-56.4), compared with 1/1107 in controls, suggesting an apparent yet overall limited contribution to predisposition to cancer. The current results also provided evidence for founder effects in the geographical distribution of these mutations. Interestingly, results from functional analysis of the breast cancer-associated ATM mutations indicated that cancer susceptibility is not restricted to mutations with dominant-negative effect on kinase activity, displayed only by 7570G>C, whereas 8734A>G showed only a partial defect in the phosphorylation of ATM substrates, and 6903insA seemed to be a null allele.

RAD50 and NBS1 are breast cancer susceptibility genes associated with genomic instability.

The Mre11 complex, composed of RAD50, NBS1 and MRE11, has an essential role in the maintenance of genomic integrity and preventing cells from malignancy. Here we report the association of three Mre11 complex mutations with hereditary breast cancer susceptibility, studied by using a case-control design with 317 consecutive, newly diagnosed Northern Finnish breast cancer patients and 1000 geographically matched healthy controls (P = 0.0004). RAD50 687delT displayed significantly elevated frequency in the studied patients (8 out of 317, OR 4.3, 95% CI 1.5-12.5, P= 0.008), which indicates that it is a relatively common low-penetrance risk allele in this cohort. Haplotype analysis and the screening of altogether 512 additional breast cancer cases from Sweden, Norway and Iceland suggest that RAD50 687delT is a Finnish founder mutation, not present in the other Nordic cohorts. The RAD50 IVS3-1G>A splicing mutation leading to translational frameshift was observed in one patient, and the NBS1 Leu150Phe missense mutation affecting a conserved residue in the functionally important BRCA1 carboxy-terminal (BRCT) domain in two patients, both being absent from 1000 controls. Microsatellite marker analysis showed that loss of the wild-type allele was not involved in the tumorigenesis in any of the studied mutation carriers, but they all showed increased genomic instability assessed by cytogenetic analysis of peripheral blood T-lymphocytes (P = 0.006). In particular, the total number of chromosomal rearrangements was significantly increased (P = 0.002). These findings suggest an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity and susceptibility to cancer.

Association of common ATM polymorphism with bilateral breast cancer.

The ATM kinase has an essential role in maintaining genomic integrity. Loss of both ATM alleles results in ataxia-telangiectasia (A-T), a rare autosomal recessive neuroimmunologic disorder associated with cancer susceptibility. Individuals heterozygous for germline ATM mutations have been reported to have an increased risk for malignancy, in particular, female breast cancer. In the current study, a full mutation analysis of the ATM gene was carried out in patients from 121 breast or breast-ovarian cancer families. We discovered that the combination of 5557G-->A in cis position with IVS38-8 T-->C was associated with bilateral breast cancer (OR = 10.2; 95% CI = 3.1-33.8; p = 0.001). As the 5557G-->A change has been reported to affect an exonic splicing enhancer, we hypothesized that the observed composite allele could have some effect on the correct splicing of exon 39. However, no aberrant transcripts were detected, but ATM expression levels of lymphoblast cell lines from heterozygous carriers of this combination allele were lower than from noncarriers (p = 0.09). Lowered gene expression levels may have direct influence on the activities in DNA damage recognition and response pathways, as well as other genome integrity maintenance functions. Based on the results, we propose a cancer risk-modifying effect for the ATM 5557G-->A, IVS38-8T-->C composite allele.

Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLC1 promoter hypermethylation.

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.