Bone morphogenetic protein 4 expression in multiple normal and tumor tissues reveals its importance beyond development.

Bone morphogenetic proteins (BMPs) are extracellular signaling molecules that belong to the transforming growth factor ß (TGFß) superfamily and are known to regulate cell proliferation, differentiation and motility, especially during development. BMP4 has an indispensable role in vertebrate development while limited information on BMP4 expression and function exists in adult tissues. Nevertheless, its contribution to cancer development and progression has gained increasing interest in recent years. Functional studies, especially in breast cancer, have implicated BMP4 both in inhibition of cell proliferation and in promotion of cell migration and invasion. To gain an insight into the function of BMP4 in normal and cancer tissues, BMP4 protein expression levels were analyzed by immunohistochemistry in 34 different normal organs/tissues, 34 different tumor types and finally in 486 breast cancer samples where possible associations between BMP4 and clinicopathological parameters were statistically evaluated. In over 20% of normal and malignant tissues, BMP4 was expressed at high level. Strong expression was observed particularly in some normal epithelial cells, such as bladder and stomach, and in squamous cell carcinomas. In breast cancer, strong BMP4 expression was detected in 25% of patients, and was associated with low proliferation index and increased frequency of tumor recurrence. Taken together, BMP4 is expressed in a subset of normal adult tissues and is likely to contribute to tissue homeostasis. However, in tumors, BMP4 expression levels vary considerably, implying diverse roles in different tumor types. This role is biphasic in breast cancer as BMP4 expression is linked to reduced proliferation and increased recurrence, thus corroborating our previous in-vitro functional data.

Androgen receptor amplification is associated with increased cell proliferation in prostate cancer.

Mechanisms of prostate cancer progression during hormonal therapy and the pathobiologic consequences of androgen receptor (AR) gene amplification are inadequately known. To further investigate the hypothesis that AR gene amplification is associated with increased cell proliferation, we analyzed 123 paraffin-embedded prostate cancer specimens from men who experienced tumor relapse during androgen withdrawal therapy. We used fluorescence in situ hybridization to quantify AR gene copy number and Ki-67 immunohistochemistry to determine cell proliferation. One third of the tumors showed AR gene amplification. Among tumors with AR amplification, the mean cell proliferation rate was 19.8 (SD, 12.3; 95% confidence interval [CI], 15.4-24.1), whereas it was 13.0 (SD, 15.9; 95% CI, 9.1-16.8) in tumors without amplification (P = .032). In the best fitting logistic regression model, only proliferation remained significant (P = .040). When the median Ki-67 labeling index (6.7%) of all tumors was used as a cutoff point, the tumors with AR amplification were more frequently highly proliferating than tumors with no amplification (P = .010; odds ratio, 3.4; 95% CI, 1.4-8.3). Our results imply that progression of prostate cancer during androgen withdrawal therapy is associated with AR gene amplification and increased cell proliferation rate in one third of tumors. We suggest that AR gene amplification is an important molecular mechanism underlying the increase in proliferation rate of a substantial fraction of recurrent prostate carcinomas. However, efforts should be targeted to develop prostate cancer cell lines to study causal relationships between AR gene amplification and various biologic variables.

A comprehensive expression survey of bone morphogenetic proteins in breast cancer highlights the importance of BMP4 and BMP7.

Bone morphogenetic proteins (BMPs) regulate diverse cellular processes, such as proliferation, differentiation, and apoptosis. The BMPs have been studied in several cancers, but thus far contradictory results have been obtained and, especially in breast cancer, information on BMPs is still limited. We performed a systematic expression survey of BMPs and their receptors in breast cancer. mRNA expression was studied of seven BMP ligands (BMP2-BMP8) and six receptors (ACVR1, BMPR1A, BMPR1B, BMPR2, ACVR2A, and ACVR2B) that specifically mediate BMP signals. Expression levels were determined in 22 breast cancer cell lines, 39 primary breast tumors, normal human mammary epithelial cell line, and normal mammary gland using semiquantitative RT-PCR. The expression frequencies and expression levels of different BMPs varied considerably in breast cancer with BMP4 and BMP7 being most frequently expressed and showing highest expression levels. The BMP specific receptors were more uniformly expressed and indicated that breast cancer is fully capable of transmitting BMP signals. Expression frequencies and levels for both the ligands and the receptors were in good concordance between the breast cancer cell lines and primary tumors. We can conclude that breast cancers possess functional BMP signaling machinery on the cell surface with distinct differences in the expression of various BMP ligands. Our survey focuses the attention particularly toward BMP4 and BMP7 and suggests their importance in breast cancer. Breast cancer cell lines and the data generated here serve as a good resource for further studies on BMP function in breast cancer.

Bone morphogenetic protein 7 is widely overexpressed in primary breast cancer.

Bone morphogenetic proteins (BMP) make up a family of extracellular signaling molecules that play a critical role in vertebrate development and both inhibit and stimulate growth in cancer cells. BMP7 was recently identified in our genomewide copy number and expression survey as being activated through amplification in breast cancer cell lines. In the present study, we further explored BMP7 gene copy number and expression changes in 22 breast cancer cell lines and 146 primary breast tumors. FISH analysis revealed that BMP7 copy number varied greatly from one cell line to another, with three cell lines showing extremely high-level amplification. Among primary tumors, BMP7 copy number was increased in 16% of the cases. BMP7 mRNA expression was determined in the cell lines and in a subset of 44 tumor samples by RT-PCR or quantitative real-time RT-PCR, respectively. Despite elevated mRNA levels in cancer cells, there was no significant association between copy number increase and mRNA expression, even though the highest expression was seen in cell lines and tumors with increased BMP7 copy number. Most interestingly, immunohistochemical analysis revealed BMP7 protein staining in all 11 breast cancer cell lines examined and strongly elevated BMP7 protein expression in 71.4% of the tumor samples as compared to normal mammary epithelium. Our results illustrate the frequent involvement of BMP7 alterations in breast cancer and especially highlight overexpression of the BMP7 protein in a very large fraction of primary breast tumors, thus suggesting a possible functional role for BMP7 in breast cancer development.

Hemochromatosis gene mutations among Finnish male breast and prostate cancer patients.

Hereditary hemochromatosis (HH), the most common genetic disease in northern Europeans, is an autosomal recessive disorder of iron metabolism. The association between hepatocellular carcinoma and HFE homozygosity is well documented, but recently HFE hetero- and homozygosity has also been linked to nonhepatocellular malignancies, including female breast cancer. We hypothesized that C282Y and H63D mutations in the HFE gene could contribute to male breast cancer (MBC) and prostate cancer (PC) susceptibility at the population level in Finland. We screened the 2 major HFE mutations, H63D and C282Y, from 116 MBC cases diagnosed in Finland between 1967 and 1996, 843 consecutive unselected PC cases diagnosed at the Pirkanmaa Hospital District between 1999 and 2001 and 480 anonymous blood donor controls by minisequencing. Our results indicate that the frequencies of the HFE mutations do not significantly differ between MBC and PC patients and the population-based controls. No significantly altered risks for MBC or PC among carriers of the 2 variants were observed. However, HFE mutations were seen twice as often among carriers of a common BRCA2 mutation 9346(-2)A-->G compared with the rest of the MBC cases, indicating that HFE may be an MBC risk modifier gene among BRCA2 mutation carriers. In conclusion, our results indicate a minor role for the HFE mutations C282Y and H63D in the causation of MBC and PC, but carriers of both BRCA2 9346(-2)A-->G and an HFE mutation may be at an increased risk.

The serine-threonine protein phosphatase PPM1D is frequently activated through amplification in aggressive primary breast tumours.

The serine-threonine protein phosphatase PPM1D is likely to play an important role in tumorigenesis. Through inactivation of p38 MAPK, PPM1D acts as a negative feedback regulator of p53 tumour suppressor gene and controls the expression of other cell cycle regulatory proteins, such as CCND1. In addition, recent knock-out mouse studies implicated PPM1D in the regulation of p16 expression and the RB tumour suppressor pathway. Here we explored the role of PPM1D aberrations in primary breast cancer. PPM1D copy number analysis showed amplification in 11% (13/117) of the tumours and quantitative real-time RT-PCR revealed a significant correlation (p = 0.0148) between PPM1D amplification and increased expression. PPM1D amplification occurred almost exclusively in tumours with wild-type p53 suggesting that these events are mutually exclusive and further confirming the role of PPM1D as a negative regulator of p53. Interestingly, PPM1D amplification was associated with ERBB2 expression (p = 0.0001) thus implying that PPM1D aberrations occurs in tumours with poor prognosis. We also explored the expression levels of two possible downstream targets of PPM1D. However, immunohistochemical analyses revealed no differences in the staining patterns of CCND1 and p16 proteins in tumours with or without PPM1D aberrations, thus suggesting that previous data from animal model experiments is not directly transferable to primary human tumours. On the other hand, these key cellular proteins are likely to be regulated through a complex fashion in breast cancer and apparently PPM1D represents only one of these mechanisms. Taken together, our findings substantiate an important role for PPM1D in breast cancer.

BRCA2 mutations in 154 finnish male breast cancer patients.

The etiology and pathogenesis of male breast cancer (MBC) are poorly known. This is due to the fact that the disease is rare, and large-scale genetic epidemiologic studies have been difficult to carry out. Here, we studied the frequency of eight recurrent Finnish BRCA2 founder mutations in a large cohort of 154 MBC patients (65% diagnosed in Finland from 1967 to 1996). Founder mutations were detected in 10 patients (6.5%), eight of whom carried the 9346(-2) A>G mutation. Two novel mutations (4075 delGT and 5808 del5) were discovered in a screening of the entire BRCA2 coding region in 34 samples. However, these mutations were not found in the rest of the 120 patients studied. Patients with positive family history of breast and/or ovarian cancer were often BRCA2 mutation carriers (44%), whereas those with no family history showed a low frequency of involvement (3.6%; P < .0001). Finally, we found only one Finnish MBC patient with 999 del5, the most common founder mutation in Finnish female breast cancer (FBC) patients, and one that explains most of the hereditary FBC and MBC cases in Iceland. The variation in BRCA2 mutation spectrum between Finnish MBC patients and FBC patients in Finland and breast cancer patients in Iceland suggests that modifying genetic and environmental factors may significantly influence the penetrance of MBC and FBC in individuals carrying germline BRCA2 mutations in some populations.

Frequent amplification and overexpression of CCND1 in male breast cancer.

Genetic events underlying the pathogenesis of breast cancer have been studied extensively and several clinically significant markers have been identified. For example, amplification and overexpression of the ERBB2 oncogene is associated with poor prognosis in breast cancer and ERBB2 serves as a target for antibody-based therapy. Current knowledge on the pathogenesis of male breast cancer (MBC) is limited. The purpose of our study was to investigate the potential relevance of a series of genes known to be amplified in female breast cancer (FBC) in a the development and pathogenesis of MBC. To this end, we applied fluorescence in situ hybridization and immunohistochemistry to the analysis of 128 breast tumors from males. Amplification of ERBB2, MYC, PPM1D and ZNF217 was detected rarely (1-2% of tumors) indicating a considerably lower amplification frequency than in FBC. CCND1 amplification was observed in 12% of cases, being in good concordance with findings from FBC. In addition, CCND1 overexpression was detected in 63% of tumors and was associated with ER positivity (p < 0.0001). Our results indicate distinct differences in the genetic basis of MBC and FBC and suggest that marked differences exist in the pathogenesis of these diseases. The lack of ERBB2 involvement was especially unexpected and implies that ERBB2-targeted therapies are unlikely to be beneficial in MBC. Furthermore, the high frequency of hormone receptor positivity and the association between ER positivity and CCND1 overexpression supports the notion that hormonal regulation is likely to be essential for the development of MBC.

CHEK2 1100delC is not a risk factor for male breast cancer population.

Genetic risk factors for male breast cancer (MBC) are poorly understood. High penetrance genes such as BRCA1 or BRCA2 account for only a small proportion of the disease. A 1100delC mutation in CHEK2 (previously known as CHK2), a cell-cycle checkpoint kinase, has been implicated in predisposition of Li-Fraumeni syndrome (LFS) and breast cancer in families suggestive of LFS. This 1100delC mutation has also been shown to confer a 2-fold increase of breast cancer risk in women and a 10-fold increase of risk in men. It was estimated to account for 1% of breast cancers in women and as much as 9% of breast cancers in men at the population level based on analysis of breast cancer families without BRCA1 or BRCA2 mutations. We wanted to evaluate the significance of CHEK2 1100delC in predisposition to MBC by assessing its frequency in a population-based material of 114 Finnish MBC patients. Two patients (1.8%) carried the 1100delC mutation. The mutation frequency among MBC cases was similar to that seen in population controls (26/1885, 1.4%). Our results indicate that CHEK2 1100delC variant does not substantially increase the risk of male breast cancer at the population level. We cannot exclude the fact that a small fraction of hereditary, family-positive male breast cancers could be attributable to CHEK2 mutations.

Amplification of a 280-kilobase core region at the ERBB2 locus leads to activation of two hypothetical proteins in breast cancer.

Amplification of the ERBB2 oncogene at 17q12 is clinically the most relevant genetic aberration in breast cancer and several studies have linked ERBB2 activation to poor clinical outcome. The development of targeted antibody-based therapy for ERBB2-overexpressing tumors and the possible role of ERBB2 as a predictor of chemotherapy treatment response have further emphasized the essential role of ERBB2 in breast cancer. Here, we performed a detailed characterization of the molecular events occurring at the ERBB2 amplicon in primary breast tumors. Analysis of the amplicon structure in 330 breast tumors by fluorescence in situ hybridization to a tissue microarray revealed a 280-kb common region of amplification that contains 10 transcribed sequences, including eight known genes. The expression levels of these 10 transcripts were determined in 36 frozen samples of grade-matched ERBB2-amplified and -nonamplified (as determined by fluorescence in situ hybridization) primary breast tumors by using quantitativereal-time reverse transcriptase-polymerase chain reaction. A highly significant association between amplification and expression levels was observed for six of these genes, including ERBB2 and two uncharacterized hypothetical proteins, MGC9753 and MGC14832. These results support the recent findings on the influence of copy number on gene expression levels and highlight novel genes that might contribute to the clinical behavior of ERBB2-amplified breast tumors.

Androgen receptor gene alterations in Finnish male breast cancer.

Mutations in the androgen receptor (AR) gene have been suggested to predispose to male breast cancer (MBC). Studies on MBC patients have not been based on the mutation screening of the entire coding region of the AR and the number of subjects has been small. Therefore, some AR gene alterations may have remained undetected. In the present study, we have comprehensively screened the entire coding region of the AR gene for mutations and also studied the role of AR CAG and GGC repeat lengths as risk factors for MBC in a cohort of 32 Finnish MBC patients. To estimate the possible involvement of the prostate cancer predisposing AR Arg726Leu germ-line mutation in MBC, this mutation was tested in 117 MBC patients. No germ-line mutations were found and the CAG and GGC repeat lengths were similar among MBC cases as among Scandinavian population. Our data indicate that the AR gene does not substantially contribute to MBC predisposition.