Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility.

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/ß-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3'-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.

A cost comparison of two strategies for treating stage IB2 cervical cancer.

Patients with stage IB2 cervical cancer at our institution are treated primarily with definitive chemoradiation, or chemoradiation followed by adjuvant hysterectomy. We sought to compare the cost differences associated with these two strategies. We identified all patients with stage IB2 cervical cancer who received their entire treatment regimen at our institution between 1995 and 2004. All patients received a combination of chemotherapy, external beam radiation, and one brachytherapy procedure, followed by either a second brachytherapy procedure or a simple hysterectomy. We retrieved cost data associated with hospitalization for the completion of respective treatment, including pharmacy, laboratory and pathology, radiation, and operating room services, as well as the costs of supplies and room and board. We identified 46 patients with stage IB2 cervical cancer, 23 who received a second brachytherapy procedure and 23 who underwent simple hysterectomy. Patients displayed similar demographics and similar disease characteristics including initial tumor diameter and histology. The cost of care for adjuvant hysterectomy group was greater ($8,316.70 vs 5,508.70, P < 0.0001). Specific differences included higher operating room costs ($1520 vs 414, P < 0.0001), pharmacy costs ($675 vs 342, P < 0.0001), and laboratory/pathology costs ($597 vs 89, P < 0.0001). We conclude that definitive chemoradiation appears to be associated with lower costs for management of stage IB2 cervical cancer when compared to simple adjuvant hysterectomy.

Chemoradiation with and without adjuvant extrafascial hysterectomy for IB2 cervical carcinoma.

The optimal treatment strategy for stage IB2 cervical carcinoma that maximizes survival while minimizing toxicity remains controversial. The purpose of this study was to compare survival and toxicity in stage IB2 cervical cancer patients treated with chemoradiation and adjuvant extrafascial hysterectomy (cRT + H) versus definitive chemoradiation (cRT). Data were abstracted from patients with IB2 cervical carcinoma primarily treated at a single institution from January 1994 to December 2004. All patients received chemotherapy concurrent with external beam radiation therapy. Patients were subsequently treated with either a single low-dose rate brachytherapy applicator followed by adjuvant extrafascial hysterectomy (n = 24) or a second brachytherapy application to complete full-dose definitive chemoradiation (n = 30). Analyses were conducted using Kaplan-Meier survival and Chi-square statistics. Groups did not differ demographically with the exception of smoking. Smokers were significantly (P = 0.04) more likely to have been treated with definitive chemoradiation. Median tumor size was similar between groups. There was no difference in overall or disease-free survival between patients who received cRT + H versus cRT (P = 0.82 and 0.75, respectively). All recurrences in the cRT arm were in smokers. There were two grade 3-4 toxicities in each group. No treatment-related deaths occurred. In this small retrospective cohort study, we observed no difference in survival between patients treated with cRT + H versus cRT. These data complement published results of Gynecologic Oncology Group studies in patients with IB2 cervical cancer. Definitive comparison between the two treatment strategies would require a randomized prospective trial with stratification based on smoking.

Well-differentiated endometrial adenocarcinomas and poorly differentiated mixed mullerian tumors have altered ER and PR isoform expression.

Both the estrogen receptor (ER) and the progesterone receptor (PR) have two subtypes: ER-alpha and beta, and PR-A and -B, respectively. These subtypes differ in function and expression, and recent reports have correlated changes in the normal proportions of these isoforms with neoplastic states. We investigated ER and PR isoform expression in normal pre- and post-menopausal endometrium, well-differentiated endometrial adenocarcinoma, and poorly differentiated malignant mixed mullerian tumors (MMMTs). Semi-quantitative RT-PCR and immunoblotting were used to measure receptor mRNA and protein expression. Estrogen receptor-alpha/beta mRNA ratios were significantly higher in postmenopausal (27.3) compared to premenopausal endometrium (4.9) mainly as a result of lower ER-beta expression in the former. Compared to age-matched postmenopausal controls, the ER-alpha/beta ratio was reduced in both grade I adenocarcinoma and MMMT specimens (3.3 and 6.8, respectively), due to a selective loss of ER-alpha. The relative abundance of PR-A and PR-B mRNA remained unchanged between all tissue subtypes. Total PR protein, however, was significantly reduced in MMMTs compared to all other groups. Thus, sex steroid receptor expression is significantly and differentially altered in well-differentiated and poorly-differentiated endometrial cancers. Both cancers exhibit decreased ER-alpha expression and the MMMTs also demonstrate a significant loss of PR protein.

Localization transition in incommensurate non-Hermitian systems.

A class of one-dimensional lattice models with an incommensurate complex potential V(theta)=2[lambda(r) cos(theta)+i(lambda)(i) sin(theta)] is found to exhibit a localization transition at /lambda(r)/+/lambda(i)/=1. This transition from extended to localized states manifests itself in the behavior of the complex eigenspectum. In the extended phase, states with real eigenenergies have a finite measure, and this measure goes to zero in the localized phase. Furthermore, all extended states exhibit real spectra provided /lambda(r)/>or=/lambda(i)/. Another interesting feature of the system is the fact that the imaginary part of the spectrum is sensitive to the boundary conditions only at the onset to localization.

Selective loss of estrogen receptor beta in malignant human colon.

Epidemiological data suggest a protective effect for estrogen replacement therapy on colon cancer. The estrogen receptor (ER) is required for the action of estrogen. The ER-beta isoform is functionally similar to ER-alpha but has a distinct pattern of expression and transcriptional response to selective estrogen response modulators. Our goal was to investigate the presence of ER-alpha and ER-beta in normal and malignant colon tissue. Human colon cancer tissue and adjacent normal colon tissue were harvested from five male and six female patients undergoing segmental colon resection for colon cancer. Western blot analysis revealed very low levels of ER-alpha protein in tumor and normal colon tissue. In both male and female patients, malignant colon tissue showed a selective loss of ER-beta protein expression when compared to normal colon tissue in the same patient. Semiquantitative reverse transcription-PCR revealed no difference in ER-beta mRNA levels between normal and malignant colon tissue. Malignant transformation of the colon is associated with a marked diminution of ER-beta protein expression, possibly through a posttranscriptional mechanism.

Expression of estrogen receptor alpha mRNA and protein variants in human endometrial carcinoma.

Breast cancer tissue has been shown to contain alternatively spliced estrogen receptor alpha (ER-alpha) mRNA variants, which have altered biological activities compared to the full-length ER-alpha. The development of endometrial cancer, as well as drug resistance in breast cancer patients undergoing tamoxifen therapy, may represent altered ER-alpha function secondary to specific exon deletions. While the literature is replete with ER mRNA variant data, little information is available regarding the presence and function of endometrial ER variant proteins. We evaluated the presence of human ER-alpha mRNA and protein variants in six premenopausal, six postmenopausal, and six endometrial carcinoma samples. Reverse transcription-polymerase chain reaction, DNA hybridization, and sequencing techniques identified exon 4, exon 5, and exon 7 mRNA splice variants in all patients as well as MCF-7 and Ishikawa cell lines. Presence of translated proteins for full-length ER-alpha, as well as splice variants, was investigated by Western blot analysis using antibodies directed against the N-terminus, hinge region, and C-terminus portions of the ER. These experiments confirmed the presence of immunopositive protein bands of approximately 64-66 kDa in all patients corresponding to wild-type ER-alpha. A protein band migrating at 41 kDa, consistent with an exon 5 splice variant, was only seen in endometrial adenocarcinoma samples. Premenopausal and postmenopausal endometrial samples did not contain detectable amounts of ER splice variant protein. Human ER-alpha mRNA variants are present in all human endometrial samples, but detectable levels of variant proteins are only observed in patients with endometrial adenocarcinoma.

Estrogen receptor mRNA splice variants in pre- and postmenopausal human endometrium and endometrial carcinoma.

Breast cancer tissue has been shown to contain alternatively spliced estrogen receptor (ER) mRNA variants which may result in alternate ER proteins. These ER variants lack specific functional domains and may alter breast cancer cells responses to both estrogen and antiestrogens. Specifically, ER variants might play a role in Tamoxifen resistance in breast cancer patients, as well as the development of endometrial carcinoma, an estrogen-dependent tumor, in patients taking this medication. We investigated the presence of ER variants in normal human endometrium and endometrial carcinoma. Ribonuclease protection assays (RPA) demonstrated ER mRNA variants in the DNA and hormone-binding domains. The reverse transcription-polymerase chain reaction (RT-PCR) assay was used to examine the ER complementary DNA (cDNA) from 25 patients, and generated two major products in both the exon 2 to 5 and 4 to 8 regions. Southern blot analysis of PCR products revealed exon 4 and 7 deletions in all 25 endometria without any qualitative differences in variant expression among premenopausal, postmenopausal, and adenocarcinoma samples. Cloning and sequencing of cDNA variants definitively identified exact deletions of either exon 4 or exon 7. These results demonstrate significant levels of ER mRNA splice variants as well as full-length ER mRNA in normal and neoplastic endometria.

The development of serotonergic projections to the olfactory bulb of Monodelphis domestica (the grey, short-tailed opossum).

The offspring of Monodelphis domestica (grey, short-tailed opossum) are born in a very immature state after a short (14-day) gestation period. As a result, they provide a useful mammalian model for examining quite early stages of brain maturation. The present study demonstrates that the onset and development of serotonergic (5-HT) projections to the Monodelphis olfactory bulb can be traced entirely after birth. 5-HT was visualized in tissue sections from postnatal day 5 (P5), P10, P20, P30, and adult opossums using avidin-biotin peroxidase immunocytochemistry. 5-HT afferent fibers are rare in the bulb until P10. Maturation occurs slowly, but by P30, 5-HT fibers assume the adult form characterized by preferential glomerular innervation. Due to the postnatal development, slow maturation, and specificity of innervation patterns, the Monodelphis bulb provides a model with which to study the regulation of serotonergic development.

Olfactory bulb organization and development in Monodelphis domestica (grey short-tailed opossum).

The olfactory bulbs of adult and developing Monodelphis domestica were examined with a number of techniques. Golgi, Nissl, and Timm stains as well as acetylcholinesterase histochemistry revealed a high degree of order within the adult bulb. All major cell classes characteristic of most mammalian species were observed. Tufted cells appeared to be restricted to the superficial portion of the external plexiform layer. Developing Monodelphis pups were examined with Nissl-stained semithin sections and with immunocytochemistry for tyrosine hydroxylase, microtubule-associated protein 2, vimentin, and glial fibrillary acidic protein. Newborn pups are extremely immature, with few postmitotic cells present in the forebrain. Considerable maturation occurs over the first four postnatal weeks, and by postnatal day 30, the bulb assumes an adult-like organization. The extreme immaturity of the bulb at birth, coupled with its strict organization, suggest that Monodelphis is a particularly appropriate species for experimental examinations of olfactory system development.