Presence Of Progesterone Receptors Research Articles

OR22-05 Progesterone Receptor-Grb2 Interaction Is Associated With Better Outcomes In Breast Cancer

Abstract Disclosure: N. Wittayavimol: None. E. Iwabuchi: None. P. Pateetin: None. Y. Miki: None. Y. Onodera: None. H. Sasano: None. V. Boonyaratanakornkit: None. The presence of progesterone receptor (PR) is usually associated with better clinical outcomes for several endocrine-related cancers. However, the molecular mechanism of how PR mediates growth inhibition remains unclear. In addition to its role as a nuclear transcription, PR also possesses an extranuclear function, mediated mainly through PR polyproline domain (PPD) interaction with the SH3 domains of cytoplasmic signaling molecules. Our previous study suggested a possible role of PR-PPD in mediating SH3-domain interaction. We showed that PR-PPD and SH3 domain interaction inhibited EGF-mediated signaling and decreased lung cancer cell proliferation. An essential adapter protein that helps transduce growth factor signaling is Grb2 which has an SH2 domain flanked by two SH3 domains. In this study, we hypothesized that PR interferes with cytoplasmic signaling through interaction between PR-PPD and Grb2-SH3. We used several protein-protein interaction assays in vitro and in cells to demonstrate PR-Grb2 interaction. GST-pulldown analysis showed that PR-PPD interacts specifically with SH3 domains of Grb2. No interaction was detected when key prolines in the PR-PPD to alanines. Immunofluorescence staining showed colocalization of PR and Grb2 in both the nucleus and cytoplasm of BT474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we showed that PR and Grb2 interacted in breast cancer cells, specifically through the Grb2-SH3 domain. No BiFC signal was detected when Grb2-SH3 domains were deleted. Furthermore, we used Proximity Ligation Assay (PLA) analysis to examine protein-protein interaction of a distance less than 40nm in cells. We found PR-Grb2 interaction in PR-positive breast cancer tissue sections. No PLA signal was detected in PR-negative breast cancer cells. Importantly, we examined 43 PR-positive breast cancer specimens and showed that PR-Grb2 interaction was associated with low histological stage and negatively correlated with lymph node invasion and metastasis of breast cancer. Our results provide a molecular mechanism for how PR can inhibit growth factor signaling by interfering with Grb2 signaling and a basis for developing novel therapeutic strategies for future cancer treatment. Presentation: Saturday, June 17, 2023

Progesterone receptors in extratesticular ducts of the Amazonian stingray Potamotrygon wallacei: A potential role in sperm maturation and aggregate formation

In cururu stingray (Potamotrygon wallacei Carvalho, Rosa and Araújo 2016) males, plasma progesterone (P4) levels appear to be associated with spermiation events. However, the specific contribution of P4 in sperm maturation via extratesticular ducts in this stingray species is unknown. With the aim of filling this knowledge gap, this study examined the morphology and the presence of progesterone receptors (PR) in the ducts, and analyzed the relationship of progesterone (P4) with sperm maturation and formation of aggregates. Morphological analysis showed that a columnar pseudostratified epithelium with stereocilia lined all the attached ducts. In active males, the secretory cells of the epididymis and the Leydig glands presented PR; however, these receptors were not found in the distal region of the epididymis (essential for nurturing and capacitation events) of regressing males. In the seminal vesicles of active males, the spermatozoa are parallelly aligned and embedded in a matrix to form the spermatozeugmata. The matrixes are formed by proteins secreted by the ducts and Sertoli cell cytoplasts. These structures presented PR, which suggests that P4 engages in sperm metabolism during storage. Our findings allude to the potential role of P4 in regulating the development and function of the attached ducts in different reproductive phases. Furthermore, P4 seems to be an essential component for regulating sperm progress, protein secretion, aggregate formation, and maintenance of sperm during storage in this freshwater stingray.

PRB inhibited cell proliferation through let-7b-E2F1 in breast cancer.

The presence of progesterone receptor (PR) and PR isoform B (PRB) in breast cancer is generally correlated with better clinical outcomes. Additionally, the significance of hormone-independent effects of PR/PRB correlated with better prognosis have been reported in non-small lung cancer (NSCLC). However, the detailed mechanism of that still remains unclear. In this study, we examined how microRNAs (miRNAs) could contribute to tumor inhibition via PR/PRB expression, in order to find miRNAs that have tumor-agnostic effects between breast cancer and NSCLC. We obtained miRNAs data using human tissues of breast cancer and NSCLC from The Cancer Genome Atlas database and PCR array from NSCLC patients of our cohort. Subsequently, we examined the function of the miRNA through in vitro study using breast cancer cell lines. As a result, only let-7b expression was significantly correlated with PR expression in both cancers. Additionally, the expression of let-7b significantly inhibited cell proliferation by inducing PR and PRB expression in breast cancer cell lines. However, the positive correlation of let-7b and PRB required a mediated factor, E2F1, obtained from TGCA databases analysis. In vitro experiments showed that let-7b significantly inhibited E2F1, and E2F1 significantly inhibited PRB. This study revealed that PRB inhibits the proliferation of breast cancer cells by the let-7b-E2F1 interaction. In addition, the immunohistochemical analysis in NSCLC was also consistent with these in vitro data. Our results could contribute to developing novel therapeutic strategies for patients with PR/PRB-positive cancer by targeting let7b or PRB expression in breast cancer and possibly NSCLC.

Mifepristone Directly Disrupts Mouse Embryonic Development in Terms of Cellular Proliferation and Maturation In Vitro.

Mifepristone (RU-486), a synthetic steroid with potent antiprogestogen and anti-glucocorticoid properties, has been widely used in clinical practice. Its effect on the endometrium, ovary, and fallopian tube has been well reported in many human and animal studies. However, its direct impact on post-implantation embryos remains underexplored. Additionally, some women choose to keep their pregnancy after mifepristone treatment fails. Thus, the potential risk remains controversial. Hence, this study investigated the direct effects of mifepristone on the development of mice blastocysts in vitro in terms of implantation and post-implantation. We detected the level of progesterone (P4) associated with ovulation in vivo. The presence of progesterone receptors (PRs) in blastocysts and post-implantation embryos was also evaluated. Cultured embryos were treated directly with mifepristone. We further examined embryonic implantation and post-implantation of blastocysts in vitro to evaluate the direct effects of mifepristone on embryos by the assessment of embryonic outgrowth and differential cell staining. In the oviduct lumen, the P4 level dramatically increased at 48 h and slightly decreased at 72 and 96 h following ovulation. PR was expressed in blastocysts not only in the preimplantation stage but also in the early post-implantation period. In the evaluation of developmental stages, mifepristone significantly reduced the successful ratio of developing into the late egg cylinder and the early somite stage. In addition, it further decreased the cell number of the embryos’ inner cell mass and trophectoderm. We herein provide evidence that mifepristone affects blastocyst viability directly and inhibits post-implantation embryo development in vitro. Furthermore, our data reveal a potential risk of fetus fatality and developmental problems when pregnancies are continued after mifepristone treatment fails.

Progesterone reduces cell survival in primary cultures of endometrioid ovarian cancer

BackgroundOvarian cancer is the most lethal of all gynecologic malignancies. The relationship between sexual steroids receptors and ovarian cancer progression has been largely evaluated. The presence of progesterone receptors has been associated with an increase of a disease-free period and overall survival in patients with ovarian carcinoma. In the present study, primary cultures of ovarian carcinoma obtained from 35 patients diagnosed with epithelial ovarian cancer were evaluated for cell survival after treatment with 10− 8 M of 17β-estradiol, progesterone, testosterone and dihydrotestosterone.ResultsThe results were analyzed considering histological subtypes: low grade serous, high grade serous, endometrioid and mucinous carcinoma; clear cell carcinoma was not included due to failure in obtaining successful cultures of this subtype. A significant reduction of cell survival was observed after progesterone treatment in endometrioid ovarian carcinoma. Changes were not observed in low grade serous, high grade serous and mucinous carcinoma. The effect of progesterone was related to the presence of progesterone receptor (PR), a 43% reduction in the cell number was observed in PR (+) endometrioid ovarian carcinoma.ConclusionsThis study supports the importance of progesterone and the presence of progesterone receptor in the reduction of ovarian cancer progression in the endometrioid ovarian carcinoma.

Progesterone in vitro increases growth, motility and progesterone receptor expression in third stage larvae of Toxocara canis

The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.

Neuromodulation of the luteal regression: presence of progesterone receptors in coeliac ganglion.

What is the central question of this study? The processes involved in luteal involution have not yet been clarified and, in general, have been studied only from a hormonal point of view. We investigated whether progesterone, from the coeliac ganglion through the superior ovarian nerve, is able to modify the luteal regression of late pregnancy in the rat. What is the main finding and its importance? We showed that the luteal regression might be reversed by the neural effect of progesterone and demonstrated the presence of its receptors in the coeliac ganglion. This suggests that the peripheral neural pathway, through neuron-hormone interaction, represents an additional mechanism to control luteal function in addition to the classical endocrine regulation. The corpus luteum (CL) is a transitory endocrine gland that produces progesterone (P). At the end of its useful life, it suffers a process of functional and structural regression until its complete disappearance from the ovary. To investigate whether P is able to regulate the process of luteal regression through the peripheral neural pathway, we used the coeliac ganglion (CG)-superior ovarian nerve-ovary system from rats on day21 of pregnancy. We stimulated the CG with P and analysed the functional regression through ovarian P release measured by radioimmunoassay, expression by RT-PCR and activity of luteal 3β- and 20α-hydroxysteroid dehydrogenase (anabolic and catabolic P enzymes, respectively). The luteal structural regression was evaluated through a study of apoptosis measured by TUNEL assay and the expression of apoptotic factors, such as Bcl-2, Bax, Fas and Fas ligand (FasL) by RT-PCR. To explore whether the effects mediated by P on the CL may be associated with P receptors, their presence in the CG was investigated by immunohistochemistry. In the group stimulated with P in the CG, the ovarian P release and the 3β-hydroxysteroid dehydrogenase activity increased, whereas the expression and activity of 20α-hydroxysteroid dehydrogenase decreased. In addition, a decrease in the number of apoptotic nuclei and a decrease of the expression of FasL were observed. We demonstrated the presence of P receptors in the CG. Overall, our results suggest that the regression of the CL of late pregnancy may be reprogrammed through the peripheral neural pathway, and this effect might be mediated by P bound to its receptor in the CG.

Abstract P3-07-04: Impact of estrogen receptor (ER) and HER2 co-expression on breast cancer characteristics and disease presentation

Abstract Background: ER and HER2 expression can alter breast cancer biology and behavior. Rationalizing that ER-negative/HER2-negative breast cancer is the more undifferentiated form of the disease we evaluated the impact of ER expression, HER2 expression, and then their co-expression on biologic and clinical characteristics of breast cancer. Methods: We analyzed a prospectively collected data set for patients diagnosed with breast cancer at the Markey Cancer Center between January 2007 and December 2012. Out of 1257 patients with a new diagnosis of invasive breast cancer, 1187 had complete ER and HER2 biomarker data and were included in this study. Among the different phenotypic groups we compared traditional patient and disease characteristics including age, tumor size, stage, grade, nodal status, and sites of metastatic disease. Statistical significance was determined using the Chi-square test, Fisher's exact test, two-sample t-test, or log-rank test as appropriate. Results: The impact of ER expression was evaluated by comparing the ER-positive/HER2-negative group (n = 762) to the ER-negative/HER2-negative breast cancer group (n = 231). ER presence was significantly associated with older age (58 vs. 53 years, p&amp;lt;0.0001), smaller mean tumor size (1.8 vs. 2.3 cm, p = 0.0003), lower grade (p&amp;lt;0.0001), earlier stage at presentation (p = 0.0002), and higher frequency of bone involvement in stage 4 disease (56.3% vs. 34.1%, p = 0.0162. There was no significant difference detected in frequency of other sites of metastatic disease. In contrast to the striking impact of ER presence, HER2 had no significant impact on any of the above parameters tested in ER-negative disease when examined by comparing the ER-negative/HER2-positive group (n = 87) to the ER-negative/HER2-negative breast cancer group. To study the impact of both ER and HER2 co-expression, we compared the ER-positive/HER2-positive breast cancer group (n = 107) to the ER-negative/HER2-negative breast cancer group. Compared to ER-negative/HER2-negative breast cancer, ER-positive/HER2-positive disease was more likely to be lower in grade (p&amp;lt; 0.0001), had higher risk of node positive status (51.1% vs. 36.8%, p = 0.0215) and had higher frequency of bone involvement in stage IV disease (62.5% vs. 34.1%, p = 0.0484). There were no significant differences in any of the other parameters tested. Interestingly, within the subgroup of ER-positive/HER2-positive disease, the presence of progesterone receptor (PgR) positivity (n = 73), which is considered indicative of more ER dependence, was somewhat unexpectedly associated with a younger age at time of presentation (50.85 vs.56.06 years, P = 0.0226) compared to PgR-negative disease (n = 34), with no other significant impact noted for PgR presence. Conclusion: When compared to ER-negative/HER2-negative breast cancer as the control group, ER presence is associated with more profound changes in key breast cancer characteristics than HER2 positivity. In contrast, when both ER and Her2 are co-expressed, the influence of ER is markedly attenuated by HER2, with a more dominant aggressive disease phenotype, albeit with retention of some ER features as reflected by lower grade and higher frequency of bone involvement in stage IV disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-04.

P5-11-11: Automated Quantification Methods Improve the Accuracy of PR as an Independent Prognostic Factor in Tamoxifen Treated Breast Cancer Patients.

Abstract Background Adjuvant endocrine therapy reduces the risk of recurrence and death in hormone receptor positive breast cancer patients. However, 40–50% of estrogen receptor (ER) positive tumors are resistant to endocrine therapy. We have previously shown that quantitative measurement of ER protein expression has limited value as a prognostic marker in tamoxifen treated patients. The presence of progesterone receptor (PR) expression has shown promise as prognostic and/or predictive marker for endocrine therapy, including adjuvant tamoxifen, but reports are contradictory. Automation of scoring methods will improve the accuracy of PR scoring and its value as a prognostic factor. Methods: This retrospective study evaluated breast cancer patients treated with adjuvant tamoxifen (n=458) from 1985–2000 at the Tom Baker Cancer Centre, Calgary, Canada. Tissue microarrays were assembled from formalin fixed paraffin embedded tumor tissue. Clinico-pathologic data was obtained from chart review. Five-year disease-free survival (DFS) was the primary outcome. DAB-based PR staining was used to generate Allred PR scores, as well as to generate scores for PR percent area expression and PR integrated optical density (PR IOD) using the DAKO ACIS III scanner and image analysis software. PR tumor nuclear pixel intensity density scores were obtained using fluorescence-based PR staining, scanned and quantified with a HistoRx PM2000 scanner andAQUA image analysis software. Continuous variables were dichotomized using Xtile. Results: In our tamoxifen cohort, 5-year DFS was associated with tumor grade [HR 4.9(3.5−6.9), p&amp;lt;0.001], tumor size [HR 2.7(1.9−3.8), p&amp;lt;0.001], lymph node status [HR 5.7(4.0−8.0), p&amp;lt;0.001] and ER status [HR 2.4(1.3−4.7), p=0.008]. Low PR was associated with significantly worse DFS regardless of the method used to quantify its expression. Methods of analysis requiring less subjective input had stronger associations between PR expression and outcome. The subjective identification of tumor and subjective biomarker quantification used in Allred scoring [HR 2.2(1.4−3.3), p&amp;lt;0.001] was less effective than ACIS scoring [PR percent area: HR 2.8(1.8−4.3), p&amp;lt;0.001; PR IOD: HR 2.9(1.9−4.6), p&amp;lt;0.001], which only requires subjective identification of the tumor. All methods were inferior to fluorescence-based AQUA scoring [HR 4.2(2.7−6.5), p&amp;lt;0.001], which automatically detects the tumor nuclear area using pan-cytokeratin and DAPI staining and measures the PR expression within this compartment. Multivariate analysis, which included age, tumor size, tumor grade, lymph node status, and ER status, confirmed that AQUA [HR 3.3(2.0−5.6), p&amp;lt;0.001] was superior to ACIS PR percent area [HR 2.3(1.4−3.9), p=0.002] and ACIS PR IOD [HR 2.7(1.6−4.8), p&amp;lt;0.001], all of which were superior to Allred scoring [HR 1.2(0.7−2.1), p=0.511]. Conclusions: We conclude that: 1) PR is an independent prognostic marker in our cohort, 2) using less subjective, automated quantitative scoring methods improves the value of PR as a prognostic biomarker, 3) AQUA appears superior to other automated methods and that 4) the incorporation of digital image analysis into practice would improve the prognostic value of PR expression in the clinical setting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-11.

Nuclear Progesterone Receptors Are Up-Regulated by Estrogens in Neurons and Radial Glial Progenitors in the Brain of Zebrafish

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation.

Depressive Symptom-Related Factors in Patients with Breast Cancer

Purpose: Patients with breast cancer are at risk for developing psychological distress and psychiatric disorders such as depression. The aim of this study was to assess the depressive symptom related factors in women with breast cancer. Methods: A total of 211 patients with breast cancer who had undergone an operation within 5 years participated in this survey with consent. Depressive symptoms in patients were measured using the 21-item Beck Depression Inventory (BDI) questionnaire in the outpatient clinic and sociodemographic, clinicopathologic factors of the patients were analyzed with a BDI score. The patients were divided into group A (BDI<16, n=146) and group B (BDI ≥16, n=65). Results: The mean age of the patients was 49.2 years and the mean follow-up period was 22 months. In the univariate analyses, postmenopausal status (p=0.014), tumor size (p=0.023) and presence of progesterone receptor (p=0.012) were significantly associated with depressive symptom. In multivariate analysis, only postmenopause (p=0.021) and tumor size (p= 0.018) were related with depressive symptom. Conclusion: Depressive symptom is common in patients with breast cancer and a screening test for depression is needed in breast cancer patient who experienced menopause or has bigger tumor size.

Finally! A Model for Progesterone Receptor Action in Normal Human Breast

Substantial clinical and experimental data over the years have supported the conclusion that estradiol-17β is the ovarian hormone primarily responsible for growth and progression of breast cancer (1). Progesterone was assumed to have negligible effects on breast tumorigenesis, in part because of its well established role to inhibit the proliferative effect of estrogen in the epithelial compartments of the endometrium and promote differentiation of the uterine glands (2). However, much publicized observational and clinical studies have more recently implicated progesterone as a proliferative hormone in the normal human breast and as a life-long risk factor for breast cancer, suggesting progesterone also contributes to early stages of breast tumorigenesis (3,4,5,6,7,8,9). Paradoxically, in established breast cancers, the presence of progesterone receptor (PR) in the primary tumor is an independent marker of favorable prognosis and is associated with a more differentiated tumor phenotype, suggesting progesterone has a protective role against progression and invasion once a tumor has developed (10,11,12). Indeed, in most PR-positive breast cancer cell lines, progestins induce a transient proliferation followed by cell cycle arrest such that the long-term effect is growth inhibition (13). This apparent dichotomy suggests that progesterone signaling is altered during disease progression. Uncertainties concerning the role of progesterone in the normal human breast and tumorigenesis have been due in part to the absence of a suitable PR-positive progesterone-responsive experimental system for the normal human breast.

Combined effects of MPA and EBRT in stage Ic endometrial cancer patients

To report the comparative effect of combined medroxyprogesterone acetate (MPA) and external beam pelvic radiotherapy (EBRT) with EBRT alone on local or distant recurrences, overall survival and treatment related toxicities in patients with stage Ic grade 3 endometrial cancer. A retrospective review of 80 International Federation of Gynecology and Obstetrics (FIGO) stage Ic grade 3 endometrial carcinoma patients treated between October 1994 and October 2004 at Renmin Hospital, Wuhan University, China was performed. All patients underwent surgery, of which 40 patients in arm I received combined MPA and EBRT while in arm II 40 patients received only adjuvant EBRT after surgery. The median dose of EBRT in arm I was 50 Gy (range 36–54 Gy) and in arm II was 45.2 Gy (range 43.2–50.4 Gy). Multivariate analysis was performed for the prognostic factors and Kaplan-Meier method was used for overall survival. Of the 80 eligible patients, 40 in each group could be evaluated. The follow-up times ranged from 4–98 months with a median of 45 months. The overall survival rates at five years were 73% among patients treated with combined MPA and EBRT and 28.2% among patients treated with EBRT alone (P < 0.001). The rate of distant metastasis was significantly higher among patients treated with EBRT alone group than combined MPA and EBRT (55% vs 25%, P = 0.006) while no difference in loco regional recurrence rates was observed in both treatment groups. Most of the side effects observed in the combined MPA and EBRT group. Age (P < 0.001) and the presence of progesterone receptors (P = 0.003) were independent significant prognostic factors for overall survival in multiple regression analysis. We has been concluded that the addition of progestagen to external beam pelvic radiotherapy significantly improved survival and reduced distant metastasis among women with stage Ic grade 3 endometrial cancer.

Solid and Pseudopapillary Tumor of the Pancreas—Review and New Insights Into Pathogenesis

Solid pseudopapillary tumors (SPT) of the pancreas are rare neoplasms that occur mostly in young women. Despite of a low malignant potential, 10% to 15% of the cases have aggressive behavior with metastatic dissemination possibly leading to death. To date, no pathological factor can reliably predict the outcome of these tumours. Galectin-3, a major actor in the carcinogenesis of pancreatic ductal adenocarcinoma, has not been investigated in SPT. The presence of progesterone receptors is frequently reported in SPT, whereas that of estrogen receptor (ER) is unclear. We studied 5 cases of SPT consisting of 4 pancreatic tumors and 1 metastatic case. The morphological distinctive feature of metastatic nodules was the presence of polygonal or spindle cells with pleiomorphic nuclei and high mitotic count exhibiting a diffuse, infiltrative growth pattern. We found a strong expression of galectin-3 in all SPTs, whereas, interestingly, it was lower in metastatic nodules. Conversely, no galectin-3 expression was found in normal pancreatic endocrine cells or in neuroendocrine tumors. We suggest therefore that galectin-3 is a useful marker to distinguish SPT from neuroendocrine tumor, and also indicator of behavior because its low expression is associated with metastatic spreading. Moreover, the presence of galectin-3 in both SPT and pancreatic ducts rises the hypothesis of a posible ductal origin of these tumors. Specific antibodies for anti-ERalpha and anti-ERbeta demonstrated a strong expression of ERbeta whereas ERalpha was not detected. In conclusion, the present study brings the first evidence of the involvement of galectin-3 in SPT but also brought up clues which allowed to reconcile previously conflicting results on the presence of ER.

Breast cancer proteomics by laser capture microdissection, sample pooling, 54‐cm IPG IEF, and differential iodine radioisotope detection

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.

Expression of female sex hormone receptors in human middle-ear cholesteatomas

The purpose of this study was to establish the eventual presence of progesterone receptor (PGR) and oestrogen receptor (EGR) in human middle-ear cholesteatoma (MECh) tissues and to compare their expression between male and female patients. An immunohistochemical technique was employed for detection of PGR- and EGR-specific immunoreactivity in MECh samples using formalin-fixed paraffin-embedded tissue sections. The positive results were verified with reverse transcriptase polymerase chain reaction (RT-PCR). The morphological study revealed stable expression of PGR in suprabasal layers of all cholesteatoma samples. Weaker immunoreactivity for PGR was demonstrated in external auditory canal skin (EACS) samples in comparison with MECh, while PGR-specific staining was not observed in retroauricular skin (RAS) samples. EGR was detected only at mRNA levels. Stronger expression of EGR PCR products was disclosed in female cholesteatoma samples, while PGR mRNA was predominantly detected in male cholesteatoma specimens. Our preliminary experimental results give us ground to assume that female sex hormones may stimulate proliferation and affect differentiation of MECh keratinocytes.

Mutational, epigenetic and expressional analyses of caveolin-1 gene in breast cancers.

Caveolin-1, an integral membrane protein of caveolae found in many cell types, has been suggested as a candidate tumor suppressor. However, the mechanism underlying caveolin-1 decreased expression is not fully understood. The purpose of this study is to investigate the role of aberrant promoter methylation in the regulation of caveolin-1 gene in breast cancer correlated with clinical findings. We used methylation specific PCR, direct sequencing and immunohistochemistry stain methods to explore the role of caveolin-1 gene in the development of breast cancer. We demonstrated that 14 of 55 cases (25.5%) and 4 of 55 cases (7.3%) had methylated CpG-island on caveolin-1 promoter in cancerous and non-cancerous cells, respectively. The frequency of aberrant promoter methylation of breast cancer tissues was significant higher than non-cancerous tissues (p<0.05). There were four types of methylation pattern of caveolin-1 gene in the breast cancer tissues. No mutation but one polymorphism GAC right curved arrow GAT at codon 82 was found in the whole exonic sequences of caveolin-1 gene. The methylation status of caveolin-1 gene had no clear relationship with age, cell grade, stage of tumor, and status of estrogen receptor, p53 and c-erbB2 in the breast cancer tissues. However, in breast tissue with aberrant promoter methylation of caveolin-1 gene, the presence of progesterone receptor showed borderline statistic difference compared to unmethylated promoter (p=0.11). Immunohistochemistry demonstrated that expression of caveolin-1 gene correlated with aberrant promoter methylation status in sporadic breast cancer tissues. Our findings suggest that aberrant promoter methylation of caveolin-1 gene is associated with inactivation of expression. This process occurs in the precancerous stage and may play an important role in the development of breast cancer.

Mouse Mammary Tumor-Like Virus Is Associated with p53 Nuclear Accumulation and Progesterone Receptor Positivity but not Estrogen Positivity in Human Female Breast Cancer

The purpose is to compare the presence of proteins with known associations with breast cancer-progesterone receptor (PgR), estrogen receptor, and p53, with the prevalence of mouse mammary tumor virus (MMTV)-like DNA sequences in human female breast cancers. A cohort of 128 Australian female breast cancers were screened for MMTV-like DNA sequences using PCR. The presence of PgR, estrogen receptor, and nuclear accumulation of p53 protein was assessed in the same samples using immunohistochemical staining. Nuclear accumulation of p53 was significantly more prevalent (P = 0.05) in archival human breast cancers containing MMTV-like DNA sequences. The presence of progesterone receptor was significantly higher in MMTV-positive than MMTV-negative breast cancers (P = 0.01). No correlation between estrogen receptor and MMTV-like DNA sequences was found. MMTV causes breast cancer in mice, and hormones up-regulate expression of virus in mice mammary tissue. It is unknown if this is the case in human breast cancers shown to contain DNA of MMTV-like viruses. The positive association between MMTV-like DNA sequences and PgR indicates hormones and MMTV may play a role in human breast cancer. Mutations of the tumor suppressor gene p53 are common in human breast cancer and are associated with higher grades of cancer. The association of MMTV-like DNA sequences with higher grades of cancer, and the positive association between p53 and MMTV-like DNA sequences clearly warrant additional investigation.

Radiation-induced meningiomas: clinical, pathological, cytokinetic, and cytogenetic characteristics.

Radiation-induced meningiomas are known to occur after high- and low-dose cranial radiation therapy. The goal of this study was to discern the distinguishing findings and characteristics of radiation-induced meningiomas. The records of 16 patients (seven men and nine women) who fulfilled the criteria for radiation-induced meningiomas were retrospectively reviewed. Clinical, histopathological, cytokinetic, and cytogenetic findings as well as the patients' outcome were analyzed. The mean age of the patients was 38.8 years and the mean tumor latency was 26.5 years. Five patients had multiple meningiomas in the irradiated field. The recurrence rate was 100% after the initial resection; 62% of patients had a second recurrence and 17% had a third recurrence. Thirty-eight percent of patients had atypical or malignant histopathological findings. The presence of progesterone receptors and low proliferation indices in these patients did not correlate with benign tumor behavior. Cytogenetic analysis showed multiple clonal aberrations in all tumors studied. The most frequent aberrations were found on chromosomes 1p, 6q, and 22. Derivative, lost, or additional chromosome 1p was found in 89% of cases and loss or deletion on chromosome 6 was found in 67%. The age of patients at presentation with meningioma and the latency period of radiation-induced meningiomas are dose related. These tumors are more aggressive and are certain to recur, have a higher histopathological grade, and are associated with complex cytogenetic aberrations particularly involving 1p and 6q.

Simultaneous chromosome 1q gain and 16q loss is associated with steroid receptor presence and low proliferation in breast carcinoma

We applied comparative genomic hybridization (CGH) to 46 breast carcinoma samples, collected from 1993 to 1995, in order to detect chromosome 1q gains and 16q losses and to define whether samples showing both these alterations had distinct biopathologic features and different clinical outcome. A total of 22 samples (48%) had simultaneous chromosome 1q gain and 16q loss, which was always associated with other genetic changes. In total, 23 samples had various chromosome imbalances (including chromosome 1q gain independent of chromosome 16q loss and vice versa) and one sample did not show detectable alterations. Samples having chromosome 1q gain/16q loss were compared to the other samples with regard to neoplasm size, lymph-node status, histologic and nuclear grade, estrogen and progesterone receptor presence, Ki-67, pRB, Cyclin D1, Cyclin A, p53, p21 and p27 expression as detected by immunohistochemistry. The samples showing chromosome 1q gain/16q loss had high steroid hormone receptor expression (P=0.02), low cell growth fraction (Ki-67, P=0.03) and high p27 expression (P<0.001). No statistical correlation with disease-free survival and overall survival or response to hormonal therapy was found. We conclude that simultaneous chromosome 1q gain/16q loss is a frequent event in invasive breast cancer, which occurs in a subset of both intermediate- and high-grade breast carcinomas. Although the final chromosome 1q and 16q imbalances might have originated from different chromosome alterations in low- and high-grade samples, the gene–dosage effect might be important in conferring peculiar biopathologic characteristics to this subset of samples. The cytogenetic and molecular mechanisms underlying these chromosome changes deserve further investigations.