José Schneider

Nup88 mRNA overexpression is associated with high aggressiveness of breast cancer

The nuclear pore complex protein Nup88 is overexpressed in tumor cells. Immunohistochemical studies have shown that this overexpression is linked to higher aggressiveness of colorectal carcinoma and to enhanced metastatic potential of melanoma cells. However, the antibodies so far developed against Nup88 have the drawback of recognizing a number of other, up to now unspecified antigens besides Nup88. For this reason, we devised the present study on Nup88 expression at the mRNA level. RNA was extracted from fresh tumor tissue corresponding to 122 breast cancer patients. Nup88 mRNA expression was measured by means of differential RT‐PCR, standardizing against a constitutive internal control gene (β‐actin). The results were dichotomized into “high” and “low” expression levels, using the median value as cut‐off. High Nup88 mRNA expression levels correlated significantly with ductal and tubular histology (p = 0.012), histologic and nuclear grade 3 of tumors (p < 0.001), absence of hormone receptor expression (p < 0.001), expression of the c‐erb‐B2 oncogene (p < 0.001), expression of mutant p53 protein (p < 0.001), high proliferation (defined by Ki67 labeling index >20%, p < 0.001), DNA aneuploidy (p < 0.001) as well as the most important ominous clinical prognostic factor, axillary node invasion (p < 0.001). We also found an inverse correlation (p < 0.001) with expression of the H‐MAM (mammaglobin) gene, a marker of low biologic and clinical aggressiveness of breast cancer. All of these factors, without exception, define a highly aggressive tumor phenotype. These findings appear to be specific to Nup88 and not to nuclear pore proteins in general. Indeed, analysis of Nup107 (which is a limiting component of the nuclear pore complex) under the same conditions in the same tumors did not yield comparable results.

Positive correlation between the expression of X-chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer

In a recent report, it has been postulated that the ubiquitous RBM proteins might constitute a novel family of apoptosis modulators. We measured the expression of the X‐chromosome RBM genes (RBMX, RBM3, and RBM10) in 122 breast cancers by means of differential RT‐PCR. Using the same method, we also studied the expression of the apoptosis‐related genes Bcl‐2 and Bax. Markers of hormone dependence (estrogen and progesterone receptors), proliferation (Ki67 and DNA‐ploidy), angiogenesis (VEGF and CD105), as well as oncogene (c‐erb‐B2), and tumor suppressor gene (p53) expression were also analyzed. The expression of all X‐chromosome RBM genes was significantly associated with the expression of the proapoptotic Bax gene (RBMX, P = 0.039; RBM3, P < 0.001; RBM10 large variant, P < 0.001; RBM10 small variant, P < 0.001). Furthermore, the expression of both RBM10 variants was significantly associated with the expression of the VEGF gene (large variant, P = 0.004; small variant, P = 0.003). We also found an association of borderline significance (P = 0.05) between the expression of RBM3, the large variant of RBM10 and wild‐type p53. Expression of the small RBM10 variant, finally, was associated with high proliferation of the tumors (Ki67 ≥ 20%; P = 0.037). The expression of both RBM10 variants seems to be interdependent to a significant degree (r = 0.26, P = 0.006). From these results, it seems that the X‐chromosome, through its RBM genes, plays a formerly unknown role in the regulation of programmed cell death (apoptosis) in breast cancer. J. Cell. Biochem. 97: 1275–1282, 2006.

Histologic Grade and CD44 Are Independent Predictors of Axillary Lymph Node Invasion in Early (T1) Breast Cancer

Background: The detection rate of small, often subclinical breast cancers is increasing in affluent societies. Concomitantly, the demand for more conservative surgical approaches is also increasing among the women affected. Predictors of the absence of nodal invasion would spare many patients with early breast cancer the risks, costs and side effects of lymphadenectomy, and thus treatment with curative intent would be applied using really minimal surgery. Patients and Methods: We reviewed the records of 135 patients with unifocal invasive breast cancers, 2 cm or less in diameter, operated upon at ‘Fundación Tejerina-Centro de Patología de la Mama’, Madrid, Spain, between January 1993 and December 1997. Full clinical and pathological data were available for all of them, together with estrogen and progesterone receptor determinations, which had been routinely performed in 134 and 133 cases, respectively. Additionally, Ki67, c-erb-B2, p53, nm23, HSP27, HSP60 and CD44std expression was studied on archival, paraffin-embedded tumor material from these same patients by means of immunohistochemistry. Results: In the univariate analysis, only histologic grade 3 (p < 0.001), Ki67 expression in more than 10% of tumor cells (p = 0.005) and CD44std negativity (p = 0.004) were significantly associated with axillary node involvement. In multivariate analysis, histologic grade 3 and CD44std negativity retained statistical significance, and thus emerged as independent predictors of nodal invasion. The combination of both, furthermore, identified a subgroup in which the axillary nodes were invariably affected. Conclusion: Some pathological and molecular features of small breast cancers were able to predict nodal metastasis significantly. However, none, either alone nor combined, was able to exclude axillary node invasion completely.

Clinicopathological significance of Nup88 expression in patients with colorectal cancer

Objective: The nucleoporin Nup88 is overexpressed in a series of human malignancies, however, its clinicopathological significance has not been studied. Our aims were to analyze Nup88 expression in normal mucosa, primary tumors and metastases from colorectal cancer patients and further to identify relationships of Nup88 expression with clinicopathological and other factors. Materials and Methods: Using immunohistochemistry, we investigated Nup88 expression in 198 primary colorectal tumors, 96 normal mucosa samples and 35 lymph node metastases. Results: The results showed that the intensity of Nup88 expression increased from the normal mucosa to the primary tumors (p < 0.0001) and tended to increase from the primary tumors to the metastases (p = 0.15). Both primary tumors and metastases presented stronger expression in the invasive margin and vascular-invaded areas. Nup88 expression was positively related to distal tumor location (p = 0.01), infiltrative growth pattern (p = 0.04) and higher proliferative activity (p = 0.04) and reversely to the grade of differentiation (p = 0.02) and apoptosis (p = 0.049). Strong expression of Nup88 predicted a worse outcome in the patients with distal tumors during the follow-up period of up to 3 years (p = 0.02). Conclusions: It seems that overexpression of Nup88 was involved in the tumorigenesis and aggressiveness of colorectal cancers, and Nup88 may be used as a prognostic factor in patients with distal tumors.

Nup88 Expression in Normal Mucosa, Adenoma, Primary Adenocarcinoma and Lymph Node Metastasis in the Colorectum

Objectives: It was the aim of this study to investigate Nup88 expression in normal colorectal mucosa, adenoma, adenocarcinoma and lymph node metastasis, as well as the relationship between Nup88 expression and clinicopathological features. Methods: Nup88 expression was examined by immunohistochemistry in 84 normal mucosa samples, 32 adenomas, 181 primary adenocarcinomas, and 18 lymph node metastases from colorectal tumour patients. Results: Nup88 expression was observed in normal epithelial and tumour cells. The frequency of strong Nup88 expression was increased from normal mucosa or adenoma to primary tumour and lymph node metastasis (p < 0.0001). There was no significant difference in the expression between normal mucosa and adenoma (p = 0.41). The frequency of strong Nup88 expression was higher in ulcerated tumours (40%) than in polypoid/large fungating tumours (23%; p = 0.048). The frequency of strong Nup88 expression was significantly different among tumours with good (21%), moderate (42%) and poor differentiation (48%; p = 0.01). Nup88 expression was not related to the patients’ gender, age, tumour location, size, histological type, invasive depth, lymph node status and Dukes stage (p > 0.05). Conclusion: Our results suggest that Nup88 may play a role during the development, aggressiveness and differentiation of colorectal tumours.

CCND1- and ERBB2-gene deregulation and PTEN mutation analyses in invasive lobular carcinoma of the breast

Because of the relatively low incidence of lobular breast carcinoma, there are very few studies on the molecular characteristics of this breast cancer. In an attempt to improve its characterization, we investigated in a large collection of invasive lobular carcinomas (ILCs) the status of markers known to be involved in the better‐studied invasive ductal carcinomas (IDC). In the current study we disposed of 80 well‐characterized ILC cases. Gene amplification of cyclin D1 (CCND1) and c‐erbB2‐encoding gene (ERBB2) and expression of their gene products were studied by differential polymerase chain reaction (PCR) and immunohistochemistry, respectively. A comprehensive point mutation study of the phosphatase and tensin homolog tumor suppressor gene (PTEN) was pursued by single strand conformation polymorphism (SSCP)/sequencing analysis. The CCND1 gene was rarely amplified in ILC in spite of showing overexpression of the protein in 41% of tumors. Hence, unlike IDC, increase in gene dosage did not account for the protein excess. PTEN mutations were detected in ILC (truncating mutations) in around 2% of the tumors. Unlike IDC, ILC did not display ERBB2 overexpression and expression of the transcription factor E2F1 correlated inversely with tumor grade. The observed discrepancy in the pattern of the human oncogenes CCND1 and ERBB2, which are involved in the process of carcinogenesis of ductal tumors, appears to suggest a different molecular basis for development and progression of ILC.

FSH receptor in vitro modulation by testosterone and hCG in human luteinized granulosa cells

OBJECTIVE To investigate the effect of testosterone and hCG on FSH receptor (FSHR) protein and mRNA expression in human granulosa cells (GC) in vitro. STUDY DESIGN Experimental in vitro cell culture obtained from healthy women undergoing IVF/ICSI due to male factor infertility. Human follicular fluid samples were obtained and after cumulus-oocyte complexes were identified, fluids were pipetted onto Ficoll gradients and centrifuged for 15 min at 400 × g at room temperature. Cells at the interface were removed and plated in 24-well plates for 3 days in M-199 with 10% FBS. Cells were treated with different concentrations of testosterone and hCG. After purification, cells were labeled with specific antibodies and the protein expression of the FSHR was evaluated by flow cytometry in the GC population. Also, total RNA was extracted from confluent GC and the FSHR gene expression was evaluated by RT-PCR. RESULTS FSHR expression was modulated by treating GC in vitro at different testosterone/hCG concentrations. When compared with untreated GC, we observed a significant effect of testosterone and hCG on the expression of the FSHR at the protein level. Time course experiments confirmed that the gene expression of the FSHR peaked at 12-24h when testosterone or hCG was used as a stimulus. CONCLUSIONS Both testosterone and hCG are able to positively modulate FSHR expression at gene and protein level in human GC in vitro.

Nup88 expression is associated with myometrial invasion in endometrial carcinoma.

Background: The nuclear pore complex protein Nup88 has been shown in previous studies to be overexpressed in tumor cells and to be associated in breast cancer with all clinical and biological features defining a more aggressive phenotype. Methods: In this pilot study, Nup88-mRNA expression was studied in a series of 29 endometrial carcinomas, of which 27 belonged to the endometrioid variety, the remaining 2 being papillary serous tumors, to verify if Nup88 plays a similar role in endometrial carcinoma as the one described in breast cancer. The tumor samples were obtained directly at the operating suite and fresh frozen at the time of hysterectomy. Nup88-mRNA expression was studied in them by means of differential reverse transcriptase-polymerase chain reaction. Nup88-mRNA expression was correlated with the clinical features of the tumors. Results: A significant correlation (r = 0.41, P = 0.027) was found between growing levels of Nup88-mRNA expression and depth of myometrial invasion. There was no correlation between Nup88-mRNA expression and the other 2 available clinical parameters, that is, tumor grade (r = 0.05, P = 0.79) and surgical stage (r = −0.18, P = 0.34). Conclusions: From these results, it is concluded that Nup88 expression seems indeed to be associated with a distinct feature of tumor aggressiveness (myometrial invasion) in endometrial carcinoma and that larger studies are therefore probably worthwhile.

Circulating estradiol defines the tumor phenotype in menopausal breast cancer patients

OBJECTIVE To correlate circulating hormone levels with the clinical and biological features of the tumors in menopausal breast cancer patients. DESIGN Circulating hormone levels were measured in 161 previously untreated menopausal breast cancer patients within 72 h of their planned surgery. The obtained hormone levels were correlated with tumor size, histological and nuclear grade, histological score, axillary nodal status, DNA-ploidy and Ki67-, c-erb-B2-, p53, Bax-, VEGF- and Nup88-expression. RESULTS The only statistically significant correlations found between circulating hormone levels and all tested variables were an inverse one between estradiol and the expression of the apoptosis-associated Bax gene (p=0.009), and again an inverse correlation between estradiol and the expression of c-erb-B2 (p=0.04). When comparing hormone levels with each other, a significant correlation between estradiol and progesterone (p<0.0001), an inverse one between estradiol and FSH (p=0.04) and a direct one between LH and prolactin (p=0.001) were found. CONCLUSION Higher circulating estradiol levels in postmenopausal breast cancer patients are associated with molecular features usually defining a biologically less aggressive tumor phenotype.

The S-phase fraction of the aneuploid cell subpopulation is the biologically relevant one in aneuploid breast cancers.

Background. In the case of DNA-aneuploid tumors there are no clear guidelines as to which S-phase fraction is the more relevant one: that corresponding to either the diploid or the aneuploid population, or rather an average of both.Materials and methods. We studied 280 breast cancer specimens from previously untreated patients. Histologically, 231 were ductal infiltrating carcinomas, 30 lobular infiltrating carcinomas and 19 corresponded to other, less frequent varieties. Postsurgically, 164 cases (58.6%) were classified as T1, 87 (31.1%) as T2 and 7 as T3. The remaining 22 cases were multifocal, diffuse tumors. Flow cytometry was performed on fresh tumor tissue, and immunohistochemistry for hormone receptors, Ki67, c-erb-B2 and p53 on paraffin-embedded material.Results. In diploid tumors, a high S-phase (above the 75th percentile) correlated significantly with Ki67 expression ≥20% (p<0.0001). In aneuploid tumors, however, this was only the case for the aneuploid fraction of tumor cells (p<0.0001). A high S-phase of diploid tumors correlated directly and significantly with a high histologic grade (p=0.04), a high nuclear grade (p=0.01), tumor size (p=0.0008), and inversely with estrogen (p<0.0001) and progesterone (p<0.0001) receptor expression. In aneuploid tumors, the aneuploid tumor fraction showed a direct and significant correlation with a high histologic grade (p=0.005), a high nuclear grade (p=0.001), mutant p53 expression (p=0.0009), and inversely with estrogen (p<0.0001) and progesterone (p=0.0001) receptor expression. A high S-phase of the diploid cell fraction of aneuploid tumors, on the other hand, just showed an inverse correlation with high nuclear grade of the tumors (p=0.02), and none whatsoever with all other tested parameters.