ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors.

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases.

BACKGROUND: It was shown recently on the level of gene expression that UGT8, coding UDP-galactose:ceramide galactosyltransferase, is one of six genes whose elevated expression correlated with a significantly increased the risk of lung metastases in breast cancer patients. In this study primary tumours and their lung metastases as well as breast cancer cell lines were analysed for UGT8 expression at the protein level. METHODS: Expression of UGT8 in breast cancer tissue specimens and breast cancer cell lines was analysed using IHC, real-time PCR and Western blotting. RESULTS: Comparison of the average values of the reaction intensities (IRS scale) showed a significant difference in UGT8 expression between (1) primary and metastatic tumours (Mann-Whitney U, P<0.05), (2) tumours of malignancy grades G3 and G2 (Mann-Whitney U, P<0.01) as well as G3 and G1 (Mann-Whitney U, P<0.001) and (3) node-positive and node-negative tumours (Mann-Whitney U, P<0.001). The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients (721). Similarly, breast cancer cell lines with the 'luminal epithelial-like' phenotype did not express or weakly expressed UGT8, in contrast to malignant, 'mesenchymal-like,' cells forming metastases in nude mice. CONCLUSION: Our data suggest that UGT8 is a significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer.

Inhibition of the Wnt/beta-catenin pathway by the WWOX tumor suppressor protein.

The WWOX gene encodes a candidate tumor suppressor protein (WWOX) implicated in a variety of human diseases such as cancer. To better understand the molecular mechanisms of WWOX action, we investigated novel partners of this protein. Using the two-hybrid system and a coimmunoprecipitation assay, we observed a physical association between WWOX and the Dishevelled protein (Dvl) family signaling elements involved in the Wnt/beta-catenin pathway. We found that enforced WWOX expression inhibited, and inhibition of endogenous WWOX expression stimulated the transcriptional activity of the Wnt/beta-catenin pathway. Inhibition of endogenous WWOX expression also enhanced the effect of Wnt-3a on beta-catenin stability. Moreover, we observed the sequestration of Dvl-2 wild type and Dvl-2NESm, a mutated form of Dvl-2 predominantly localized in the nucleus, in the cytoplasm compartment by WWOX. Our results indicate that WWOX is a novel inhibitor of the Wnt/beta-catenin pathway. WWOX would act, at least in part, by preventing the nuclear import of the Dvl proteins.

Prognostic value of maspin mRNA expression in ER alpha-positive postmenopausal breast carcinomas.

Maspin, a member of the serpin family, has a role in cell migration, angiogenesis and apoptosis. Little is known of the clinical significance of maspin gene expression in human cancers. We developed a real-time quantitative RT-PCR assay to quantify the full range of maspin mRNA copy numbers in a series of 10 ER alpha-positive and 10 ER alpha-negative breast tumours. We observed a statistical link between low maspin mRNA levels and positive oestrogen status (P=0.0012). In consequence, to better assess the prognostic value of maspin gene expression in breast cancer, we then quantified maspin mRNA content in an additional independent well-defined cohort of 105 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Maspin expression varied widely in tumour tissues (by nearly four orders of magnitude), being underexpressed in 33 out of 105 tumours (31.4%) and overexpressed in 24 out of 105 tumours (22.9%) relative to normal breast tissues. Immunohistochemical studies demonstrated that maspin protein was strictly expressed in myoepithelial cells of normal breast tissue and in tumour epithelial cells, exclusively in maspin-overexpressing tumours. Patients with tumours overexpressing the maspin gene had significantly shorter relapse-free survival after surgery than patients whose tumours normally expressed or underexpressed maspin (P=0.0011). The prognostic significance of maspin overexpression persisted in Cox multivariate regression analysis (P=0.0024). These findings show that the maspin mRNA level can have important prognostic significance in human breast cancer, and point to the maspin gene as a putative molecular predictor of hormone responsiveness in breast cancer.

Somatic genetic alterations (LOH) in benign, borderline and invasive ovarian tumours: intratumoral molecular heterogeneity.

Loss of heterozygosity (LOH) affects a number of chromosome regions in ovarian cancer, pointing to the possible involvement of tumour-suppressor genes in ovarian tumorigenesis. We performed comparative analysis of allelic loss at 6 frequently affected chromosome regions in a panel of 53 benign, borderline and malignant ovarian tumours. Precursor lesions could provide evidence that an accumulation of genetic events is required for normal ovarian epithelium to generate malignant tumours. LOH on chromosome 1p was relatively common in benign, borderline and malignant tumours, while at 11p and 7q it was observed not only in invasive but also in borderline tumours. Moreover, 17q and 18q were affected mainly in advanced malignant tumours and revealed a high frequency of clonal intratumoral heterogeneity. We encountered different spectra of genetic alterations in primary tumours and their metastasis, which may be the results of intratumoral heterogeneity leading to dissemination in only some sub-clones.

Location of several putative genes possibly involved in human breast cancer progression.

Cancer is a genetic disease resulting from an accumulation of genetic abnormalities in various regulatory genes. Most studies on genetic alterations in human breast cancer have involved primary tumors. The possible involvement of specific tumor suppressor genes in the later stages of cancer progression is poorly documented. We investigated allelic losses associated with breast cancer progression by analyzing 55 polymorphic markers on 11 autosomal chromosomes in a series of 49 relapses (23 local recurrences and 26 distant metastases). All of the loss of heterozygosity (LOH) regions reported in primary breast tumors were frequent in both series of relapses. These results suggest that the allelic losses that are common to the different series of samples occur very early during tumor progression. This study points to candidate metastasis-related genes targeted by LOH on chromosome arms 3p21.3, 16q22.2-23.2, and, possibly, 7q31 but provides no clear evidence of LOH affecting previously described metastasis-related genes such as NME1, MTS1, and TSG101.

Low frequency of TSG101/CC2 gene alterations in invasive human breast cancers.

Large intragenic deletions of the TSG101/CC2 gene were recently reported in seven of 15 primary metastatic breast cancers. Although the number of samples was small, this observation suggested that TSG101/CC2 alterations were a major event in breast carcinogenesis. To study the frequency of these deletions in invasive breast cancers we analysed 189 primary invasive breast tumours and 59 breast cancer metastases. We detected intragenic rearrangements in only three samples (two primary tumours and one metastasis). Northern blot analysis of 43 tumours without rearrangements failed to detect any abnormalities. Furthermore, we studied TSG101/CC2 in 11 human breast adenocarcinoma cell lines by Southern blot, RT-PCR and sequencing of the entire coding region of the gene, and detected no abnormalities. These results show that genetic alteration of TSG101/CC2 is a rare event in breast cancer.

Loss of heterozygosity on chromosome arm 16q in breast cancer metastases.

One of the main genetic abnormalities associated with breast carcinogenesis is the loss of genetic material from chromosome arm 16q. Different groups have identified two regions (16q22.1 and 16q24-ter) that are frequently deleted in primary tumors, suggesting the presence of tumor suppressor genes in these regions. Little is known about the late stages of tumor progression in this respect, and we, therefore, analyzed biopsy specimens of breast cancer metastases for deletions in these critical regions of 16q. We examined fine needle cytopunctures from 24 metastases, each with lymphocyte DNA, for allelic imbalance on 16q by means of polymerase chain reaction (PCR) with 15 highly polymorphic markers. All the metastatic samples showed deletion of at least one informative locus on 16q. The loss of heterozygosity (LOH) pattern often indicated the loss of a complete long arm of chromosome 16 (13 cases); nevertheless, in the remaining 11 samples, partial LOH patterns were observed. A small region of overlap (SR02) in 16q22.1 was frequently involved, whereas another (SR01) in 16q24-ter was affected in only two cases. A third region of LOH in 16q22.2-q23.2 was found in 6/11 samples. These results suggest that at least three different regions are involved in allelic imbalance on chromosome arm 16q in breast cancer. Loss of material from the third region could be a major event in the genesis of metastases.

Genetic alteration mapping on chromosome 7 in primary breast cancer.

Alterations of chromosome 7 are among the most frequent cytogenetic abnormalities found in human breast carcinoma. We examined genetic changes on chromosome 7 in 113 primary human breast tumors, using both microsatellite and restriction fragment length polymorphism/variable number of tandem repeats polymorphism markers mapping to the long arm (15 markers) and the short arm (8 markers). Allelic imbalance at 1 or more loci was observed in 50 (44%) of 113 tumors on the long arm of chromosome 7 and in 41 (36%) tumors on the short arm. Genetic changes of one arm were significantly associated with alterations of the other arm. The 50 7q-altered tumor DNAs exclusively showed a loss of heterozygosity (LOH), 23 (46%) at all informative loci tested on 7q and 27 (54%) at some loci (interstitial and/or telomeric deletions on 7q). The pattern of LOH of these 27 tumors enabled us to identify 3 distinct consensus regions of deletions on 7q, only 1 of which (7q31 region) has already been described in breast cancer. Among the 41 7p-altered tumor DNAs, 32 had a gain and/or loss of the entire short arm of chromosome 7. Fourteen tumor DNAs showed an allelic gain, and 18 tumor DNAs showed a LOH at each locus on the short arm. The other 9 7p-altered tumors showing partial random alterations of chromosome 7p revealed no common altered regions. This is the first report of an association between alterations of DNA sequences on chromosome 7p and breast cancer. The results suggest that tumor suppressor genes are present on the long arm of chromosome 7 and are associated with breast tumorigenesis. Moreover, the frequent loss or gain of a whole copy of chromosome 7p suggests the involvement of a gene dosage effect of this chromosomal arm in the pathogenesis of breast cancer.

Loss of heterozygosity at chromosome 16q in prostate adenocarcinoma: identification of three independent regions.

Loss of heterozygosity (LOH) on chromosome arm 16q is one of the most consistent genetic alterations in sporadic prostate cancer and may be involved in cancer development through inactivation of tumor suppressor genes. A candidate tumor suppressor gene on this chromosome arm, CDH1 at 16q22.1, is dysregulated in prostate cancer. However, no specific deletion map has been constructed from prostate tumors to determine whether CDH1 is the potential target gene for the observed LOH on 16q. To narrow down the region of 16q loss, we constructed a detailed deletion map that incorporates CDH1. We examined the pattern of allelic imbalance in prostate tissue from 22 patients with confined prostate tumors, 22 with local extracapsular extension, and 15 with metastatic forms, using 14 CA microsatellite repeats on 16q. Thirty-five of the 59 tumors tested showed LOH for at least one marker. We found evidence of 16q monosomy in 5 cases and partial allelic loss in 30. Our data provide evidence that three different target regions on 16q might be involved in the pathogenesis of prostate cancer. The first region is telomeric and lies at 16q24.3 between markers D16S520 and D16S413; the second, the most centromeric region in the 16q22.1 band, and limited by markers D16S347 and D16S318, is close to the CDH1 gene; the third, intermediate region, at 16q23.2, is bracketed by loci D16S518 and D16S507. The rate of LOH at 16q24.3 was significantly higher in metastatic forms (80%; 12 of 15) than localized forms (32%; 7 of 22), pointing to a gene related to invasiveness in prostate cancer.

Classical gene amplifications in human breast cancer are not associated with distant solid metastases.

To determine the relationship between breast cancer progression and gene amplification, we screened 62 distant metastases and 122 primary breast tumours for the amplification of the proto-oncogenes MYC and ERBB2 and the 11q13 chromosomal region. Surprisingly, solid metastases showed an absence of gene amplification. These results suggest that the amplification of the proto-oncogenes MYC and ERBB2 and the 11q13 chromosomal region seem to be involved mainly in the genesis of the primary breast tumour rather than its progression.