Yesim Gökmen-Polar

Protein kinase Cι is required for Ras transformation and colon carcinogenesis in vivo

Protein kinase C ι (PKCι) has been implicated in Ras signaling, however, a role for PKCι in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCι is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCι in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCι (kdPKCι) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCι in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCι. Our data demonstrate that PKCι is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCι, and Rac1.

Mapping of a molecular determinant for protein kinase C β(II) isozyme function

In human erythroleukemia (K562) cells, the highly related protein kinase C (PKC) α and PKC βIIisozymes serve distinct functions in cellular differentiation and proliferation, respectively. Previous studies using two domain switch PKC chimera revealed that the catalytic domains of PKC α and βII contain molecular determinants important for isozyme-specific function (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9156–9160). We have now analyzed a panel of PKC chimeras to determine the specific region within the catalytic domain important for PKC βII function. A cellular assay for PKC βII function was devised based on the finding that PKC βII selectively translocates to the nucleus and phosphorylates nuclear lamin B in response to the PKC activator bryostatin. This response is strictly dependent upon expression of PKC βII or a PKC chimera that functions like PKC βII. We demonstrate that a PKC α/βIIchimera containing only the carboxyl-terminal 13 amino acids from PKC βII (βII V5) is capable of nuclear translocation and lamin B phosphorylation. These results are consistent with our recent observation that the PKC βII V5 region binds to phosphatidylglycerol (PG), a potent and selective PKC βII activator present in the nuclear membrane (Murray, N. R., and Fields, A. P. (1998) J. Biol. Chem. 273, 11514–11520). Soluble βII V5 peptide selectively inhibits PG-stimulated PKC βII activity in a dose-dependent fashion, indicating that PG-mediated activation of PKC βII involves interactions with the βII V5 region of the enzyme. We conclude that βII V5 is a major determinant for PKC βIInuclear function and suggest a model in which binding of PG to the βII V5 region stimulates nuclear PKC βIIactivity during G2 phase of the cell cycle.

Extrathoracic metastases of thymic origin: a review of 35 cases.

Thymic tumors are categorized as types A, AB, B1, B2, B3, and thymic carcinoma under the World Health Organization (WHO) classification. Thymomas are typically slow growing tumors that predominantly involve the surrounding structures through direct invasion, while thymic carcinomas tend to be more aggressive. A significant number of patients are asymptomatic and can present with metastases as the first presentation. The exact incidence of extrathoracic metastases from thymoma is not known. This study describes a series of 35 cases of histologically documented metastatic thymomas and thymic carcinomas at extrathoracic sites. These cases were classified according to the current World Health Organization (WHO) classification criteria, and we present their clinical data as well as discuss the differential diagnoses of these lesions. Our study shows that all types of thymic tumors, regardless of histologic type, can be associated with invasion and metastases to thoracic and extrathoracic sites.

Prognostic Impact of HOTAIR Expression is Restricted to ER-Negative Breast Cancers

Expression of HOX transcript antisense intergenic RNA (HOTAIR), a large intergenic noncoding RNA (lincRNA), has been described as a metastases-associated lincRNA in various cancers including breast, liver and colon cancer cancers. We sought to determine if expression of HOTAIR could be used as a surrogate for assessing nodal metastases and evaluated RNA in situ hybridization (RNA-ISH) assay in a tissue microarray constructed from 133 breast cancer patients. The prognostic value of HOTAIR was further validated in large cohorts using The Cancer Genome Atlas (TCGA) breast cancer subjects. RNA-ISH analysis was successful in 94 cases (17% cases scored 0, 32.9% scored 1, 30.8% scored 2, and 19.1% scored 3). The expression of HOTAIR did not correlate with nodal metastasis regardless of the scoring intensity or with other study parameters (age, tumor size and grade, expression status). Further analysis of TCGA dataset showed that HOTAIR expression was lower in ductal carcinomas but higher in ER-negative tumors. Overexpression of HOTAIR was not associated with nodal metastases or prognosis in ER-positive patients. Its function as a poor prognostic indicator in ER-negative patients was restricted to node-positive patients. HOTAIR appears to be a marker for lymphatic metastases rather than hematogenous metastases in ER-negative patients.

2-methoxyestradiol inhibits the anaphase-promoting complex and protein translation in human breast cancer cells.

2-methoxyestradiol (2ME2), an estradiol metabolite with antiproliferative and antiangiogenic activities, is in phase I/II clinical trials for breast cancer. 2ME2 inhibits microtubule polymerization and causes cells to arrest in G2-M. The purpose of this study was to further elucidate the molecular mechanism of 2ME2. MDA-MB-435 breast cancer cells were treated with 2ME2 (2 micromol/L) or vehicle alone. RNA was extracted and genomic profiling was done using 22k Agilent microarrays. Expression Analysis Systematic Explorer was used to determine enrichment of Gene Ontology categories. Protein isolates were subjected to Western blot analysis. Protein synthesis was measured with a [35S]methionine pulse assay. An MDA-MB-435 cell line with two beta-tubulin mutations (2ME2R) was used to determine whether novel mechanisms were tubulin-dependent. Gene Ontology categories enriched include genes that regulate the mitotic spindle assembly checkpoint, apoptosis, and the cytosolic ribosome. The target of the mitotic spindle assembly checkpoint is the anaphase-promoting complex (APC). APC inhibition was confirmed by measuring protein levels of its targets securin and cyclin B1, which were increased in 2ME2-treated cells. Because gene expression in the cytosolic ribosome category was decreased, we evaluated whether 2ME2 decreases protein translation. This was confirmed with a pulse assay, which showed decreased isotope incorporation in 2ME2-treated cells, which was maintained in the tubulin-resistant 2ME2R cells. APC inhibition was not maintained in 2ME2R cells. 2ME2 induces tubulin-dependent cell cycle arrest through regulation of genes involved in the mitotic spindle assembly checkpoint, which results in inhibition of the APC and tubulin-independent inhibition of protein translation.

The role of histology in predicting recurrence of type A thymomas: a clinicopathologic correlation of 23 cases

Spindle cell thymomas (type A), as per the WHO definition, are benign tumors with an excellent prognosis. However, recent studies document aggressive behavior with local recurrences as well as extrathoracic metastases. More recently, Nonaka and Rosai have raised the question as to whether atypical features like cellular atypia, mitotic activity, necrosis, and vascular permeation could predict the adverse outcomes of these tumors. In an effort to address the ‘atypia and outcome’ issue of spindle cell thymomas, we analyzed our database of over 600 cases of thymic tumors to identify type A thymomas. The presence of histomorphological features like tumor size, nuclear shape and variability, mitotic rate, and presence/absence of necrosis were correlated with Masaoka stage, relapse/recurrence, and extrathoracic metastases. The study identified 23 patients of pure spindle cell thymomas (WHO type A) ranging in age from 40 to 88 years (median age, 54 years) and with male-to-female ratio of 1:0.9. Approximately 43% of the cases had recurrence or metastases during the followup period (average, 49 months). The presence of necrosis correlates with both relapse and extrathoracic metastases but not with the stage of diagnosis. However, none of the other clinical or histological features, including size, predominant nuclear shape, nuclear variability, and mitotic activity, were correlated with the outcome parameters, such as stage at diagnosis, presence or absence of relapse, and extrathoracic metastases. Histological atypia is fairly common in WHO type A thymomas. The presence of necrosis was associated with both locoregional and systemic disease. However, none of the other clinical or histological features correlated with aggressive behavior.

A Gene Signature to Determine Metastatic Behavior in Thymomas

Purpose Thymoma represents one of the rarest of all malignancies. Stage and completeness of resection have been used to ascertain postoperative therapeutic strategies albeit with limited prognostic accuracy. A molecular classifier would be useful to improve the assessment of metastatic behaviour and optimize patient management. Methods qRT-PCR assay for 23 genes (19 test and four reference genes) was performed on multi-institutional archival primary thymomas (n = 36). Gene expression levels were used to compute a signature, classifying tumors into classes 1 and 2, corresponding to low or high likelihood for metastases. The signature was validated in an independent multi-institutional cohort of patients (n = 75). Results A nine-gene signature that can predict metastatic behavior of thymomas was developed and validated. Using radial basis machine modeling in the training set, 5-year and 10-year metastasis-free survival rates were 77% and 26% for predicted low (class 1) and high (class 2) risk of metastasis (P = 0.0047, log-rank), respectively. For the validation set, 5-year metastasis-free survival rates were 97% and 30% for predicted low- and high-risk patients (P = 0.0004, log-rank), respectively. The 5-year metastasis-free survival rates for the validation set were 49% and 41% for Masaoka stages I/II and III/IV (P = 0.0537, log-rank), respectively. In univariate and multivariate Cox models evaluating common prognostic factors for thymoma metastasis, the nine-gene signature was the only independent indicator of metastases (P = 0.036). Conclusion A nine-gene signature was established and validated which predicts the likelihood of metastasis more accurately than traditional staging. This further underscores the biologic determinants of the clinical course of thymoma and may improve patient management.

Dual targeting of EphA2 and ER restores tamoxifen sensitivity in ER/EphA2-positive breast cancer

Overexpression and altered function of EphA2 receptor tyrosine kinase are critical in the progression of breast cancer and provide a target for breast cancer therapy. We have previously demonstrated that EphA2 overexpression decreases estrogen dependence and Tamoxifen sensitivity both in vitro and in vivo. EA5, a novel monoclonal antibody that mimicks the binding of ephrin A to EphA2, reverses the effect of EphA2 overexpression and restores Tamoxifen sensitivity in EphA2-transfected MCF-7 cells in vitro. To explore the role of EphA2 overexpression on ER-dependent mechanisms, we used two different ER+/EphA2-transfected cell line models (MCF-7neo/MCF-7EphA2 and T47Dneo/T47DEphA2). EA5 inhibits primary tumor growth and restores Tamoxifen sensitivity in the MCF-7EphA2 xenografts. Using the T47DEphA2 in vitro model, we verified that EphA2 decreases ER activation in response to E2 stimulation consistent with our earlier results in MCF-7EphA2 model. We found no direct interaction between ER and EphA2 and no difference in expression of canonical ER-dependent proteins or ER co-regulators. However, E2 stimulation phosphorylates FAKTyr925 only in ER+/EphA2+ cell lines. Treatment of T47DEphA2 cells with EA5 and Tamoxifen leads to dephosphorylation of FAKTyr925 in response to E2. Our data demonstrate that dual targeting of EphA2 and ER is a promising approach for delaying resistance to Tamoxifen. The data support our hypothesis that EphA2 impacts ER function via a FAK dependent pathway.

Biomarkers for breast cancer stem cells: the challenges ahead

Recent studies suggest that a subset of cancer cells with the ability for self-renewal and differentiation into different cell lineages is responsible for tumor progression, metastasis and resistance therapy. These cells, designated as tumor-initiating cells, tumor-propagating cells or cancer stem cells, are of great interest for cancer prognostication and therapeutics. Numerous cell surface and intracellular markers exhibiting cancer stem cell characteristics have been identified in breast cancer, presenting a promise to use them as biomarkers. However, there is a great need for the improvement of experimental methods to detect them in clinical samples, and validate their utility as predictors of the disease outcome, propensity for metastasis and response to treatment. In this article, we present an overview of the current status of breast cancer stem cells, with a focus on biomarkers. We also discuss the technical challenges on the road to defining breast cancer stem cells as biomarkers.

Expression levels of SF3B3 correlate with prognosis and endocrine resistance in estrogen receptor-positive breast cancer

De novo or acquired resistance to endocrine therapy limits its utility in a significant number of estrogen receptor-positive (ER-positive) breast cancers. It is crucial to identify novel targets for therapeutic intervention and improve the success of endocrine therapies. Splicing factor 3b, subunit 1 (SF3B1) mutations are described in luminal breast cancer albeit in low frequency. In this study, we evaluated the role of SF3B1 and SF3B3, critical parts of the SF3b splicing complex, in ER-positive endocrine resistance. To ascertain the role of SF3B1/SF3B3 in endocrine resistance, their expression levels were evaluated in ER-positive/endocrine-resistant cell lines (MCF-7/LCC2 and MCF-7/LCC9) using a real-time quantitative reverse transcription PCR (qRT-PCR). To further determine their clinical relevance, expression analysis was performed in a cohort of 60 paraffin-embedded ER-positive, node-negative breast carcinomas with low, intermediate, and high Oncotype DX recurrence scores. Expression levels of SF3B1 and SF3B3 and their prognostic value were validated in large cohorts using publicly available gene expression data sets including The Cancer Genome Atlas. SF3B1 and SF3B3 levels were significantly increased in ERα-positive cells with acquired tamoxifen (MCF-7/LCC2; both P<0.0002) and fulvestrant/tamoxifen resistance (MCF-7/LCC9; P=0.008 for SF3B1 and P=0.0006 for SF3B3). Expression levels of both MCF-7/LCC2 and MCF-7/LCC9 were not affected by additional treatments with E2 and/or tamoxifen. Furthermore, qRT-PCR analysis confirmed that SF3B3 expression is significantly upregulated in Oncotype DX high-risk groups when compared with low risk (P=0.019). Similarly, in publicly available breast cancer gene expression data sets, overexpression of SF3B3, but not SF3B1, was significantly correlated with overall survival. Furthermore, the correlation was significant in ER-positive, but not in ER-negative tumors.This is the first study to document the role of SF3B3 in endocrine resistance and prognosis in ER-positive breast cancer. Potential strategies for therapeutic targeting of the splicing mechanism(s) need to be evaluated.