Juan C. Díaz-Chico

Androgens and androgen receptors in breast cancer.

Aromatase (CYP19) converts adrenal and ovarian androgens into estrogens, which supports the growth of estrogen-dependent breast cancers. Anti-aromatase agents are displacing antiestrogens as the first-line treatment for estrogen receptor positive breast cancers. Androgens can act as estrogen precursors, but besides this capability they can also directly act on breast cancer cells by binding to androgen receptors, which are present in the majority of breast cancer specimens. Epidemiological and clinical evidences suggest that higher levels of circulating androgen increase the risk of developing breast cancer. Androgen receptor gene polymorphisms which render the more transcriptionally active receptors have been related to a lower risk of breast cancer. It is currently accepted that androgens act as antiproliferative agents in the presence of estrogens in some breast cancer cell lines. However, emerging evidence suggests that direct androgenic activity might also stimulate cell growth in a subset of estrogen-resistant breast tumors. Here we discuss the supporting evidence which proposes that androgens themselves are actively involved in breast carcinogenesis and its clinical behaviour.

The relationship between microsatellite instability and PTEN gene mutations in endometrial cancer

Microsatellite instability (MSI) and mutations in the PTEN gene are among the molecular alterations involved in endometrial carcinogenesis. There is conflicting information regarding to their role in this type of tumor. For this reason, we have studied both molecular lesions in a large population‐based series of 205 patients with sporadic endometrial cancer. MSI was found in 41 (20.0%) of the tumors and PTEN mutations were found in 74 (36.1%). There were differences in genotype between tumors with and without MSI. Tumors with MSI showed both a higher frequency of PTEN mutations (58.5% vs. 30.4%) (p = 0.002, Fishers exact test) and a higher number of insertions or deletions (I/D) of one nucleotide within the mononucleotide tracts of the PTEN gene (45.8% vs. 11.4% out of all I/D, p = 0.005). Conversely, G:C to A:T transitions in CpG dinucleotides were found mostly in microsatellite stable tumors (57.7% vs. 18.2% out of all single‐base substitutions, p = 0.037). Overall, 67.6% of tumors with mutated PTEN exhibited multiple mutations or allelic imbalance (AI). Multiple PTEN mutations in the same tumor were more frequent in tumors with MSI (60% vs. 25.7%); by contrast the presence of AI accompanying PTEN mutation was higher in microsatellite stable tumors (74.3% vs. 40%) (p = 0.028). In addition, patients with both genetic alterations were diagnosed at more advanced stage of progression (54.2% for MSI vs. 20.0% for MSS, p = 0.006), and exhibited a worse prognosis (hazard ratio [95% confidence interval]: 3.0 [1.1–13.1], p = 0.034, log‐rank test) than patients with only the PTEN gene mutated. Our data suggest that the DNA mismatch repair system status influences: (i) both the frequency and the mutational spectrum of PTEN; (ii) the nature of one of the hits that inactivate this tumor‐suppressor gene; and (iii) the clinical condition and behavior of the patients.

Endometrial stromal sarcoma expression of estrogen receptors, progesterone receptors and estrogen-induced srp27 (24K) suggests hormone responsiveness.

The endometrial stroma plays a decisive role in sustaining the gland epithelium along the menstrual cycle, and in preparing the microenvironment that allows embryo implantation. The stroma undergoes important changes during the menstrual cycle that affects both the cell number and differentiation. These changes are regulated by both estrogen and progesterone. Stromal sarcomas are extremely rare, occurring much less than any other uterine tumor. Their origin and biology are poorly understood. The purpose of this work was to try to learn more about the stromal physiology, and also to ascertain whether the stromal sarcoma has characteristics of hormone dependence. We studied the presence of estrogen receptors (ER), progesterone receptors (PR) and the stress-responsive protein of 27K (srp27, a protein first described as an estrogen-induced 24K protein in MCF-7 cells) in both normal stroma and stromal sarcoma. The ER and PR were measured by exchange assays. The srp 27 was studied both by Western-blot and by IHC by means of specific monoclonal antibodies. The stromal sarcomas studied showed a high concentration of both ER (96 to 116 fmol/mg prot.) and PR (565 to 995 fmol/mg prot.). These amounts of ER and PR were higher than the mean found in normal endometrium during the proliferative phase (43 and 637 fmol/mg prot., respectively), and much higher than that of the secretory phase (17 and 229 fmol/mg prot., respectively). The srp27 characterized by Western-blot in both the normal stroma and stromal sarcoma was found to be similar to the srp27 of breast cancer. The IHC results showed a very low expression of srp27 in the stroma during the proliferative phase that increases when the endometrium enters the secretory phase. The low-malignancy grade stromal sarcomas showed abundant expression of srp27, but the high-malignancy grade sarcomas showed no expression of srp27. The obtained results prove the stroma capability to express the srp27. A negative correlation between malignancy of stromal tumors and srp27 expression was found. The presence of ER and PR in some stromal sarcomas proves that they have characteristics of hormone responsiveness. These findings suggest that ER and PR assays should be routinely performed in stromal sarcomas as well as in endometrial adenocarcinomas, and also that antiestrogenic drugs might be considered for the treatment of ER and PR positive stromal sarcomas.

Alleles with short CAG and GGN repeats in the androgen receptor gene are associated with benign endometrial cancer.

The human androgen receptor (AR) gene possesses 2 trinucleotide repeats of CAG and GGN in exon 1. The CAG repeat corresponds to a polyglutamine tract in the N‐terminal region of the receptor, that affects its transcriptional efficiency. The GGN repeat codifies for a polyglycine tract, and affects the amount of the AR protein transcribed. The endometrium contains ARs and the androgens have antiproliferative properties in cultured endometrial cancer (EC) cells. Larger CAG repeats of the AR gene give rise to a weaker transcriptional activity and have been found to be associated with endometrial carcinogenesis. The possible involvement of CAG and GGN tracts in the progression of EC is unknown. To study that possibility, we have genotyped both CAG and GGN polymorphisms of the AR gene in tumor tissue genomic DNA from a series of 204 consecutive patients with EC, and analyzed the results with regard to the pathological features and clinical outcome of patients. We classified the alleles as S (short ≤ median; S‐CAG ≤21 repeats; S‐GGN ≤22 repeats) or L (long > median). The genotype with both S‐CAG repeat alleles (SS‐CAG) was more common in patients diagnosed at an early stage (41.6% SS‐CAG vs 22.6% SL‐ and LL‐CAG together, p = 0.048) and in tumors that did not invade the vascular space (43.0% SS‐CAG vs 26.4% SL‐ and LL‐CAG together, p = 0.034). The genotype with SS‐GGN alleles was more common in well‐differentiated tumors (41.2% SS‐GGN vs 25.2% LS‐ and LL‐GGN together, p = 0.017) and in endometrioid histological subtype tumors (35.3% SS‐GGN vs 13.0% SL‐ and LL‐GGN together, p = 0.034). When the genotypes of both repeats coexisting in each tumor specimen were taken into consideration, the relationship between the SS‐CAG genotype and early stage remained only in the presence of the SS‐GGN genotype (43.9% vs 0%, p = 0.01). No other associations were observed. In univariate survival analysis, patients with short alleles of both repeats (SS‐CAG and SS‐GGN genotypes simultaneously) had a lower risk of cancer‐specific death (p = 0.032, mean follow‐up: 63 months). Our data suggests that short CAG or GGN repeats of the AR gene are associated with a more benign condition of traditional prognostic variables in EC.

Double strand break repair components are frequent targets of microsatellite instability in endometrial cancer

AIM DNA double strand break (DSB) repair is a central cellular mechanism of the DNA damage response to maintain genomic stability. DSB components are frequently mutated in colorectal cancer with microsatellite instability (MSI). We investigated whether DSB repair is involved in endometrial cancer (EC) with MSI. METHODS Mononucleotide microsatellite tracts of 14 genes of the DSB repair system were analysed in a series of 41 EC with MSI. Among these genes, the microcephalin 1 (MCPH1/BRIT1) has never been tested as target of MSI in tumour series. RESULTS The most frequently mutated gene was DNAPKcs (n=14, 34%) followed by RAD50 (n=7, 17%), MRE11, ATR and BRCA1 (n=6, 15%), and by CtIP and MCPH1 (n=5, 12%). While DSB biallelic mutations were infrequent, a high proportion of tumours (n=30, 73%) presented mutations at some component of the DSB repair pathway, and almost half of them showed alterations at two or more components. Tumours with mutations in two or more genes were significantly associated with advanced grade (p=0.03) and vascular invasion (p=0.02) and marginally associated with advanced stage (p=0.07). CONCLUSIONS Our results suggest that in EC, the DSB repair is a relatively common mutational target of MSI and might contribute to tumour progression, and also that MCHP1 may be a novel target gene of MSI.

Microsatellite Instability Predicts Clinical Outcome in Radiation-Treated Endometrioid Endometrial Cancer

PURPOSE To elucidate whether microsatellite instability (MSI) predicts clinical outcome in radiation-treated endometrioid endometrial cancer (EEC). METHODS AND MATERIALS A consecutive series of 93 patients with EEC treated with extrafascial hysterectomy and postoperative radiotherapy was studied. The median clinical follow-up of patients was 138 months, with a maximum of 232 months. Five quasimonomorphic mononucleotide markers (BAT-25, BAT-26, NR21, NR24, and NR27) were used for MSI classification. RESULTS Twenty-five patients (22%) were classified as MSI. Both in the whole series and in early stages (I and II), univariate analysis showed a significant association between MSI and poorer 10-year local disease-free survival, disease-free survival, and cancer-specific survival. In multivariate analysis, MSI was excluded from the final regression model in the whole series, but in early stages MSI provided additional significant predictive information independent of traditional prognostic and predictive factors (age, stage, grade, and vascular invasion) for disease-free survival (hazard ratio [HR] 3.25, 95% confidence interval [CI] 1.01-10.49; p = 0.048) and cancer-specific survival (HR 4.20, 95% CI 1.23-14.35; p = 0.022) and was marginally significant for local disease-free survival (HR 3.54, 95% CI 0.93-13.46; p = 0.064). CONCLUSIONS These results suggest that MSI may predict radiotherapy response in early-stage EEC.

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

HER‐2/neu oncogene status and total cellular p185HER‐2 content were simultaneously analyzed in 415 invasive breast‐cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut‐off value of 1.7 for the ratio between the intensity of the HER‐2/neu gene band and the reference gene band, to consider the HER‐2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER‐2 over‐expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER‐2/neu gene amplification. Of these tumors, 87% showed over‐expression of the p185HER‐2. Of the remaining 352 specimens that did not display HER‐2/neu gene amplification, 97% showed no p185HER‐2 over‐expression (p < 0.0001). In 40 selected samples with a p185HER‐2 level lower than 260 fmol/mg protein, the degree of p185HER‐2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER‐2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER‐2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER‐2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER‐2/neu oncogene is amplified or p185HER‐2 is over‐expressed.

Estrogens regulate the hepatic effects of growth hormone, a hormonal interplay with multiple fates.

The liver responds to estrogens and growth hormone (GH) which are critical regulators of body growth, gender-related hepatic functions, and intermediate metabolism. The effects of estrogens on liver can be direct, through the direct actions of hepatic ER, or indirect, which include the crosstalk with endocrine, metabolic, and sex-differentiated functions of GH. Most previous studies have been focused on the influence of estrogens on pituitary GH secretion, which has a great impact on hepatic transcriptional regulation. However, there is strong evidence that estrogens can influence the GH-regulated endocrine and metabolic functions in the human liver by acting at the level of GHR-STAT5 signaling pathway. This crosstalk is relevant because the widespread exposition of estrogen or estrogen-related compounds in human. Therefore, GH or estrogen signaling deficiency as well as the influence of estrogens on GH biology can cause a dramatic impact in liver physiology during mammalian development and in adulthood. In this review, we will summarize the current status of the influence of estrogen on GH actions in liver. A better understanding of estrogen-GH interplay in liver will lead to improved therapy of children with growth disorders and of adults with GH deficiency.

The GGN and CAG repeat polymorphisms in the exon-1 of the androgen receptor gene are, respectively, associated with insulin resistance in men and with dyslipidemia in women

The human androgen receptor (AR) gene possesses two trinucleotide repeats of CAG and GGN in exon-1. The GGN repeat affects the amount of AR protein translated, while the CAG repeat affects the efficiency of AR transcriptionaly. In this study, we have genotyped these polymorphic tracts in a representative sample of 557 Caucasian adult individuals (314 women and 243 men) from the Canary Islands, Spain (the ENCA Study), and investigated their association with fasting serum levels of lipids, glucose and insulin. The number of CAG repeats in women (expressed as the average length of the two alleles) was inversely correlated with serum levels of LDL-cholesterol (Spearman rho=-0.179; P<0.01). Women with an average number of CAG repeats in the upper tertile showed significantly lower levels of LDL-cholesterol than those grouped in the lower and middle tertile, after adjusting for age, body mass index, waist-to-hip ratio, smoking and alcohol drinking. The number of GGN repeats in men was correlated with fasting insulin levels (Spearman rho=-0.206; P<0.01), the homeostasis model assessment of insulin resistance (HOMA-IR; Spearman rho=-0.230; P<0.01) and the McAuley index of insulin sensitivity (Spearman rho=0.194; P<0.01). Men with a number of GGN repeats in the upper tertile showed lower levels of insulin and HOMA and a higher level of the McAuley index than those grouped in the lower and middle tertile, after adjusting for the variables listed above. These results support the hypothesis that the longer alleles of the CAG and GGN polymorphisms in the exon-1 of the AR gene, indicative of lower androgenic signaling, respectively protect women from developing dyslipemia and men from developing insulin resistance.

Quantitative analysis of p185HER‐2/neu protein in breast cancer and its association with other prognostic factors

The total cellular p185HER‐2/neu protein (p185) content was measured by ELISA in 346 invasive primary breast cancers, and the results were compared with those of estrogen (ER) and progesterone (PR) receptors, pS2 and Cathepsin D (Cat D) content. At a cut‐off level of 260 fmol/mg protein, 53 of the 346 tumors (15%) were p185‐positive. A significant positive correlation was observed between p185 levels and those of Cat D, and a weaker, though significant, positive correlation with ER, and pS2 levels, but not with those of PR. However, when only the 293 p185‐negative tumors were considered, the correlation between p185 and ER improved substantially, and statistical significance was reached for PR. p185‐positive tumors exhibited lower ER and PR content and higher Cat D content than p185‐negative tumors. The pS2 content, in contrast, did not undergo significant variation. Tumors considered to be p185‐positive were significantly more frequently positive for Cat D at the cut‐off of 45 pmol/mg protein, and were more frequently negative for ER and/or PR, but only significant at the cut‐off of 15 fmol/mg or higher for both steroid receptors. Finally, p185 status was not associated with menopausal status, tumor size, axillary‐lymph‐node invasiveness or distant metastases. These results suggest that 260 fmol/mg protein as the cut‐off for p185 allows the identification of a tumoral sub‐population with a more aggresive phenotype.Int. J. Cancer 74:175–179, 1997.