Luciane R. Cavalli

Deletion, methylation, and expression of the NKX3.1 suppressor gene in primary human prostate cancer.

NKX3.1 is a prostate-specific homeoprotein and tumor suppressor that is affected by the loss of 8p21 in prostate cancer. In mice, Nkx3.1 haploinsufficiency results in prostatic dysplasia and complements cancer formation induced by loss of other suppressor genes. However, NKX3.1 expression can be immunohistochemically detected in most primary prostate cancers. We examined the relationship between suppressor gene haploinsufficiency, methylation, and quantitative NKX3.1 expression levels in primary prostate cancer. NKX3.1 gene copy number was assessed by microsatellite analysis, fluorescence in situ hybridization, and quantitative PCR. NKX3.1 gene methylation was determined in prostate cancer cell lines and we thereby identified potential CpG methylation sites for methylation-specific PCR analysis in tissues. We validated and then applied an internally controlled fluorescence immunomicroscopic assay for NKX3.1 protein expression in 48 primary prostate cancer specimens from radical prostatectomies. NKX3.1 loss of heterozygosity was found in 27 of 43 tissues tested. Classic CpG island methylation of the NKX3.1 gene was not found in either prostate cancer cell lines or tissues. However, in 33 of 40 samples tested, CpG sites at -921, -903, and -47 were methylated to a greater degree in malignant than in adjacent normal cells. In 43 of 48 samples, NKX3.1 protein expression was reduced from 0.34 to 0.90 compared with adjacent normal luminal epithelium (mean of all samples, 0.68; 95% confidence interval, 0.05). In 12 cases that also had high-grade prostatic intraepithelial neoplasia, NKX3.1 expression levels were similar in preinvasive and invasive cancer cells and significantly lower than adjacent normal cells. Even in the presence of allelic loss, NKX3.1 expression is reduced over a wide range in prostate cancer at the time of prostatectomy, suggesting that diverse factors influence expression. Samples with protein expression below the median level in cancer cells had both NKX3.1 deletion and selective CpG methylation.

ERRγ Mediates Tamoxifen Resistance in Novel Models of Invasive Lobular Breast Cancer

One-third of all estrogen receptor (ER)-positive breast tumors treated with endocrine therapy fail to respond, and the remainder is likely to relapse in the future. Almost all data on endocrine resistance has been obtained in models of invasive ductal carcinoma (IDC). However, invasive lobular carcinomas (ILC) comprise up to 15% of newly diagnosed invasive breast cancers each year and, whereas the incidence of IDC has remained relatively constant during the last 20 years, the prevalence of ILC continues to increase among postmenopausal women. We report a new model of Tamoxifen (TAM)-resistant invasive lobular breast carcinoma cells that provides novel insights into the molecular mechanisms of endocrine resistance. SUM44 cells express ER and are sensitive to the growth inhibitory effects of antiestrogens. Selection for resistance to 4-hydroxytamoxifen led to the development of the SUM44/LCCTam cell line, which exhibits decreased expression of ERalpha and increased expression of the estrogen-related receptor gamma (ERRgamma). Knockdown of ERRgamma in SUM44/LCCTam cells by siRNA restores TAM sensitivity, and overexpression of ERRgamma blocks the growth-inhibitory effects of TAM in SUM44 and MDA-MB-134 VI lobular breast cancer cells. ERRgamma-driven transcription is also increased in SUM44/LCCTam, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity. These data support a role for ERRgamma/AP1 signaling in the development of TAM resistance and suggest that expression of ERRgamma may be a marker of poor TAM response.

Detection of LOH and mitochondrial DNA alterations in ductal lavage and nipple aspirate fluids from high-risk patients

We describe a method for the isolation of free DNA from ductal lavage (DL) and nipple aspirate fluid (NAF), and its evaluation for the presence of LOH at the BRCA1 and FHIT genes and for mitochondrial DNA (mtDNA) mutations at the D310 marker, to improve early detection of breast cancer. We evaluated 26 DL and six NAF samples from 14 women of known BRCA1 status, who have no clinical evidence of breast tumors: nine mutation carriers and five non-carriers. LOH studies at the BRCA1 locus were possible in 19/26 DL samples, and at the FHIT locus in 16/26 samples. In 4/9 mutation carriers we found LOH at the BRCA1 allele, and in two of these we also found LOH at the FHIT allele. In one of the mutation carriers with BRCA1 LOH, invasive breast cancer was subsequently detected, and the tumor showed the same LOH as the DL. In one of the true negatives, BRCA1 and FHIT LOH were detected. The mitochondrial studies were possible in all 26 DL samples and a somatic mutation was found in 3/9 carriers, two of whom also had LOH at the BRCA1 locus, and in none of the non-carriers. mtDNA mutation evaluation was possible in 4/6 NAF samples. The NAF and DL results were concordant. One NAF sample from a BRCA1 patient showed a mtDNA mutation. Our data demonstrates the feasibility of performing molecular studies using the free DNA present in the ductal fluid, while the intact cells can be used for cytologic studies.

Multicolour FISH and quantitative PCR can detect submicroscopic deletions in holoprosencephaly patients with a normal karyotype

Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain. At birth, nearly 50% of children with HPE have cytogenetic anomalies. Approximately 20% of infants with normal chromosomes have sequence mutations in one of the four main HPE genes (SHH, ZIC2, SIX3, and TGIF). The other non-syndromic forms of HPE may be due to environmental factors or mutations in other genes, or potentially due to submicroscopic deletions of HPE genes. We used two complementary assays to test for HPE associated submicroscopic deletions. Firstly, we developed a multicolour fluorescent in situ hybridisation (FISH) assay using probes for the four major HPE genes and for two candidate genes (DISP1 and FOXA2). We analysed lymphoblastoid cell lines (LCL) from 103 patients who had CNS findings of HPE, normal karyotypes, and no point mutations, and found seven microdeletions. We subsequently applied quantitative PCR to 424 HPE DNA samples, including the 103 samples studied by FISH: 339 with CNS findings of HPE, and 85 with normal CNS and characteristic HPE facial findings. Microdeletions for either SHH, ZIC2, SIX3, or TGIF were found in 16 of the 339 severe HPE cases (that is, with CNS findings; 4.7%). In contrast, no microdeletion was found in the 85 patients at the mildest end of the HPE spectrum. Based on our data, microdeletion testing should be considered as part of an evaluation of holoprosencephaly, especially in severe HPE cases.

Loss of heterozygosity in normal breast epithelial tissue and benign breast lesions in BRCA1/2 carriers with breast cancer

Loss of heterozygosity (LOH) of the wild-type BRCA1/2 allele is a reproducible event in breast tumors of BRCA1/2 mutation carriers, but it is unknown if this allelic loss occurs only in association with recognizable histopathologic abnormalities. We evaluated the early genomic changes that occur in the mammary glands of patients with increased predisposition to breast cancer due to germline mutations in the BRCA1/2 genes. We tested the hypothesis that these genomic changes may be detected, not only in histologically abnormal and malignant breast tissues, but also in morphologically normal tissues and in areas with pathologically benign changes. Samples were obtained from five breast cancer patients: four BRCA1 carriers and one BRCA2 carrier. In each case, nontumor tissue areas surrounding the tumor or from other locations of the breast were isolated using laser capture microdissection. We evaluated 29 areas showing normal terminal ductal lobular units (TDLUs) or histopathologically benign changes (in particular, sclerosing adenosis), using a panel of polymorphic dinucleotide microsatellite markers for the BRCA1 gene and other chromosome 17 loci, for the BRCA2 gene and other chromosome 13 loci, and for the FHIT gene on 3p14.2. Overall, we analyzed a total of 105 samples of nontumor tissues; LOH was detected in 59 of the 105 (56%). In the normal TDLUs, 15 of 30 samples (50%) showed LOH; in the tissues with benign proliferative changes, such as sclerosing adenosis, 44 of 75 samples showed LOH (59%). Our results suggest that there is a field effect of early genetic events preceding morphologic changes in the mammary glands of BRCA mutation carriers.

Genetic and epigenetic alterations in sentinel lymph nodes metastatic lesions compared to their corresponding primary breast tumors

The accumulation of genetic and epigenetic changes plays a pivotal role in tumor development and progression. In this study, we investigated these changes using comparative genomic hybridization and bisulfite polymerase chain reaction analysis for CpG island hypermethylation of the following genes: TP16, THBS2, E-Cadherin (ECAD), RARbeta2, MINT1, MINT2, and MINT31 in six paired primary breast tumors and their matched sentinel lymph nodes (SLN). The most frequent chromosomal alterations observed were the following: losses of 6q13 approximately q23 and 13q13 approximately q32 and gains of 9q31 approximately qter, 11p15 approximately q21, 12q23 approximately qter, and 20q12 approximately qter. Gain of 6p21 approximately pter was observed in the SLN but in none of the primary tumors. Overall, 71% (30/42) of the methylation measurements were identical between the primary tumors and the SLN. Of the six cases, two showed no differences between the primary tumors and SLN, one tumor with 4 of 7 genes hypermethylated in the primary tumor showed loss of all four hypermethylation events in the SLN, and the remaining three tumors showed loss of one methylation event and simultaneous gain of one to two methylation changes in the SLN. This is the first study reporting genetic and epigenetic alterations in breast sentinel lymph nodes compared to their corresponding primary tumors. Characterization of such alterations may lead to identification of initial events associated with the metastatic dissemination process.

Cooperation of tumor-derived HBx mutants and p53-249ser mutant in regulating cell proliferation, anchorage-independent growth and aneuploidy in a telomerase-immortalized normal human hepatocyte-derived cell line

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53‐249ser mutation. Recently, we have reported that tumor‐derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53‐mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor‐derived HBx mutants along, or in cooperation with p53‐249ser, could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte‐specific genes. We found that overexpression one of the tumor‐derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild‐type allele CNT significantly decreased CFE in HHT4 cells. p53‐249ser rescued CNT‐mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT‐expressing HHT4 cells could form colonies, which could be significantly enhanced by p53‐249ser. Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53‐249ser. Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.

Peripheral-type benzodiazepine receptor (PBR) gene amplification in MDA-MB-231 aggressive breast cancer cells

Recent studies using human breast cancer cell lines, animal models, and human tissue biopsies have suggested a close correlation between the expression of the peripheral-type benzodiazepine receptor (PBR) and the progression of breast cancer. This study investigates the genetic status of the PBR gene in two human breast cancer cell lines: MDA-MB-231 cells, which are an aggressive breast cancer cell line that contains high levels of PBR, and MCF-7 cells, which are a nonaggressive cell line that contains low levels of PBR. Both DNA (Southern) blot and fluorescence in situ hybridization analyses indicate that the PBR gene is amplified in MDA-MB-231 relative to MCF-7 cells. These data suggest that PBR gene amplification may be an important indicator of breast cancer progression.

Androgen-regulated and highly tumorigenic human prostate cancer cell line established from a transplantable primary CWR22 tumor

Purpose: One of the major obstacles in understanding the molecular mechanisms underlying the transition of prostate cancer growth from androgen dependency to a hormone-refractory state is the lack of androgen-regulated and tumorigenic human prostate cancer cell lines. Experimental Design: We have established and characterized a new human prostate cancer cell line, CWR22Pc, derived from the primary CWR22 human prostate xenograft tumors. Results: The growth of CWR22Pc cells is induced markedly by dihydrotestosterone, and CWR22Pc cells express high levels of androgen receptor (AR) and prostate-specific antigen (PSA). Importantly, PSA expression in CWR22Pc cells is regulated by androgens. Stat5a/b, Stat3, Akt, and mitogen-activated protein kinase were constitutively active or cytokine inducible in CWR22Pc cells. The AR in CWR22Pc cells contains the H874Y mutation, but not the exon 3 duplication or other mutations. When inoculated subcutaneously into dihydrotestosterone-supplemented castrated nude mice, large tumors formed rapidly in 20 of 20 mice, whereas no tumors developed in mice without circulating dihydrotestosterone. Moreover, the serum PSA levels correlated with the tumor volumes. When androgens were withdrawn from the CWR22Pc tumors grown in nude mice, the tumors initially shrank but regrew back as androgen-independent tumors. Conclusions: This androgen-regulated and tumorigenic human prostate cancer cell line provides a valuable tool for studies on androgen regulation of prostate cancer cells and on the molecular mechanisms taking place in growth promotion of prostate cancer when androgens are withdrawn from the growth environment. CWR22Pc cells also provide a model system for studies on the regulation of transcriptional activity of mutated H874YAR in a prostate cancer cell context.

Molecular markers of breast axillary lymph node metastasis.

In breast cancer, axillary lymph node status is one of the most important prognostic variables and a crucial component to the staging system. Several clinico-histopathological parameters are considered to be strong predictors of metastasis; however, they fail to accurately classify breast tumors according to their clinical behavior and to predict which patients will have disease recurrence. Methods based on genome-wide microarray analyses have been used to identify molecular markers with respect to the development of axillary lymph node metastasis. Most of these markers can be detected in the primary tumors, which can potentially lead to the ability to identify patients at the time of diagnosis who are at high risk for lymph node metastasis, allowing for early intervention and more suitable adjuvant treatments.