COX-2 expression in operable triple negative breast cancer: A hospital based cross-sectional study

Objectives. Triple Negative Breast Cancer (TNBC) lack expression of estrogen receptors (ER), progesterone receptors (PR), and absence of Her2 gene amplification. Current literature has identified TNBC and over-expression of cyclo-oxygenase-2 (COX-2) protein in primary breast cancer to be independent markers of poor prognosis in terms of overall and distant disease free survival. The purpose of this study was to compare COX-2 over-expression in TNBC patients to those patients who expressed one or more of the three tumor markers (i.e. ER, and/or PR, and/or Her2).^ Methods. Using a secondary data analysis, a cross-sectional design was implemented to examine the association of interest. Data collected from two ongoing protocols titled "LAB04-0657: a model for COX-2 mediated bone metastasis (Specific aim 3)" and "LAB04-0698: correlation of circulating tumor cells and COX-2 expression in primary breast cancer metastasis" was used for analysis. A sample of 125 female patients was analyzed using Chi-square tests and logistic regression models. ^ Results. COX-2 over-expression was present in 33% (41/125) and 28% (35/124) patients were identified as having TNBC. TNBC status was associated with elevated COX-2 expression (OR= 3.34; 95% CI= 1.40–8.22) and high tumor grade (OR= 4.09; 95% CI= 1.58–10.82). In a multivariable analysis, TNBC status was an important predictor of COX-2 expression after adjusting for age, menopausal status, BMI, and lymph node status (OR= 3.31; 95% CI: 1.26–8.67; p=0.01).^ Conclusion. TNBC is associated with COX-2 expression—a known marker of poor prognosis in patients with operable breast cancer. Replication of these results in a study with a larger sample size, or a future randomized clinical trial demonstrating an improved prognosis with COX-2 suppression in these patients would support this hypothesis.^

Survival analysis of circulating tumor cells in triple-negative breast cancer

Background. Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008. [1] Triple-negative breast cancer (TNBC) is an aggressive phenotype comprising 10–20% of all breast cancers (BCs). [2-4] TNBCs show absence of estrogen, progesterone and HER2/neu receptors on the tumor cells. Because of the absence of these receptors, TNBCs are not candidates for targeted therapies. Circulating tumor cells (CTCs) are observed in blood of breast cancer patients even at early stages (Stage I & II) of the disease. Immunological and molecular analysis can be used to detect the presence of tumor cells in the blood (Circulating tumor cells; CTCs) of many breast cancer patients. These cells may explain relapses in early stage breast cancer patients even after adequate local control. CTC detection may be useful in identifying patients at risk for disease progression, and therapies targeting CTCs may improve outcome in patients harboring them. Methods . In this study we evaluated 80 patients with TNBC who are enrolled in a larger prospective study conducted at M D Anderson Cancer Center in order to determine whether the presence of circulating tumor cells is a significant prognostic factor in relapse free and overall survival . Patients with metastatic disease at the time of presentation were excluded from the study. CTCs were assessed using CellSearch System™ (Veridex, Raritan, NJ). CTCs were defined as nucleated cells lacking the presence of CD45 but expressing cytokeratins 8, 18 or 19. The distribution of patient and tumor characteristics was analyzed using chi square test and Fisher's exact test. Log rank test and Cox regression analysis was applied to establish the association of circulating tumor cells with relapse free and overall survival. Results. The median age of the study participants was 53years. The median duration of follow-up was 40 months. Eighty-eight percent (88%) of patients were newly diagnosed (without a previous history of breast cancer), and (60%) of patients were chemo naïve (had not received chemotherapy at the time of their blood draw for CTC analysis). Tumor characteristics such as stage (P=0.40), tumor size (P=69), sentinel nodal involvement (P=0.87), axillary lymph node involvement (P=0.13), adjuvant therapy (P=0.83), and high histological grade of tumor (P=0.26) did not predict the presence of CTCs. However, CTCs predicted worse relapse free survival (1 or more CTCs log rank P value = 0.04, at 2 or more CTCs P = 0.02 and at 3 or more CTCs P < 0.0001) and overall survival (at 1 or more CTCs log rank P value = 0.08, at 2 or more CTCs P = 0.01 and at 3 or more CTCs P = 0.0001. Conclusions. The number of circulating tumor cells predicted worse relapse free survival and overall survival in TNBC patients.^

NOVEL THERAPEUTIC TARGETS IDENTIFIED BY HIGH-THROUGHPUT TECHNOLOGIES FOR TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancers (TNBC) are characterized by the lack of or reduced expression of the estrogen and progesterone receptors, and normal expression of the human epidermal growth factor receptor 2. The lack of a well-characterized target for treatment leaves only systemic chemotherapy as the mainstay of treatment. Approximately 60-70% of patients are chemosensitive, while the remaining majority does not respond. Targeted therapies that take advantage of the unique molecular perturbations found in triple-negative breast cancer are needed. The genes that are frequently amplified or overexpressed represent potential therapeutic targets for triple-negative breast cancer. The purpose of this study was to identify and validate novel therapeutic targets for triple-negative breast cancers. 681 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in 2 data sets. For two genes, 3 of the 4 siRNAs showed preferential growth inhibition in TNBC cells. These two genes were the low density lipoprotein receptor-related protein 8 (LRP8) and very low-density lipoprotein receptor (VLDLR). Exposure to their cognate ligands, reelin and apolipoprotein E isoform 4 (ApoE4), stimulated the growth of TNBC cells in vitro. Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. High-throughput protein and metabolic arrays revealed that ApoE4 stimulation rescued TNBC cells from serum-starvation induced up-regulation of genes involved in lipid biosynthesis, increased protein expression of oncogenes involved in the MAPK/ERK and DNA repair pathways, and reduced the serum-starvation induction of biochemicals involved in oxidative stress response and glycolytic metabolism. shLRP8 MDA-MB-231 xenografts had reduced tumor volume, in comparison to parental and shCON xenografts. These results indicate that LRP8-APOE signaling confers survival advantages to TNBC tumors under reduced nutrient conditions and during cellular environmental stress. We revealed that the LRP8-APOE receptor-ligand system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro and helps to sustain the growth of MDA-MD-231 xenografts. We propose that inhibitors of LRP8-APOE signaling may be clinically useful therapeutic agents for triple-negative breast cancer.

CIP4 and Src in Promoting the Migration and Invasion of Breast Cancers

Cellular invasion represents a critical early step in the metastatic cascade, and many proteins have been identified as part of an “invasive signature.” The non-receptor tyrosine kinase Src is commonly upregulated in breast cancers, often in conjunction with overexpression of EGFR. Signaling from this pathway stimulates cell proliferation, migration, and invasion and frequently involves proteins that regulate the cytoskeleton. My data demonstrates that inhibition of Src, using the small-molecule inhibitor dasatinib, impairs cellular migration and invasion. Furthermore, Src inhibition sensitizes the cells to the effects of the chemotherapeutic doxorubicin resulting in dramatic, synergistic inhibition of proliferation with combination treatments. The Src-targeted protein CIP4 (Cdc42-interacting protein 4) associates with curved plasma membranes to scaffold complexes of Cdc42 and N-WASp. In these experiments, I show that CIP4 overexpression correlates with triple-negative biomarker status, cellular migration, and invasion of (breast cancer cells. Inhibition of CIP4 expression significantly decreases migration and invasion. Furthermore, I demonstrate the novel finding that CIP4 localizes to invadopodia, which are finger-like projections of the actin cytoskeleton that are associated with matrix degradation and cellular invasion. Depletion of CIP4 in invasive cells impairs the formation of invadopodia and the degradation of gelatin. Therefore, CIP4 is a critical component of the invasive phenotype acquired by human breast cancer cells. In this body of work, I propose a model in which CIP4 promotes actin polymerization by stabilizing the active conformation of N-WASp. CIP4 and N-WASp are both phosphorylated by Src, implicating this pathway in Src-dependent cytoskeletal rearragement. This represents a novel role for F-BAR proteins in migration and invasion.

Application of cell line based genomic predictors to predict response to targeted therapies in breast cancer

Cancer cell lines can be treated with a drug and the molecular comparison of responders and non-responders may yield potential predictors that could be tested in the clinic. It is a bioinformatics challenge to apply the cell line-derived multivariable response predictors to patients who respond to therapy. Using the gene expression data from 23 breast cancer cell lines, I developed three predictors of dasatinib sensitivity by selecting differentially expressed genes and applying different classification algorithms. The performance of these predictors on independent cell lines with known dasatinib response was tested. The predictor based on weighted voting method has the best overall performance. It correctly predicted dasatinib sensitivity in 11 out of 12 (92%) breast and 17 out of 23 (74%) lung cancer cell lines. These predictors were then applied to the gene expression data from 133 breast cancer patients in an attempt to predict how the patients might respond to dasatinib therapy. Two predictors identified 13 patients in common to be dasatinib sensitive. Sixty two percent of these cases are triple negative (ER-negative, HER2-negative and PR-negative) and 76% are double negative. The result is consistent with the findings from other studies, which identified a target population for dasatinib treatment to be triple negative or basal breast cancer subtype. In conclusion, we think that the cell line-derived dasatinib classifiers can be applied to the human patients. ^

Proliferation index in ductal carcinoma in situ of the breast: Relation to estrogen, progesterone and HER2/neu receptors

Background. Ductal carcinoma in situ (DCIS) is the most prevalent precursor to invasive breast cancer (IBC), the second leading cause of death in women in the United States. The three most important prognostic markers for IBC are Estrogen receptor (ER), Progesterone receptor (PR) and HER2/neu. The four groups (IBC) defined as (1) ER and/or PR positive and HER2/neu negative, (2) ER and/or PR positive and HER2/neu positive (3) ER and/or PR negative and HER2/neu positive and (4) negative for all three of these receptors (Triple negative). However, they have not been well studied in DCIS. This is an exploratory study with a primary objective to examine the prevalence of ER, PR, and HER2/neu in DCIS, to explore if the defined groups of IBC occur in DCIS and to consider the biological relationship between these four groups and the proliferative activity of the tumor. A secondary goal of this study is to examine the relationship between grade and proliferative activity. Methods. Using immunohistochemistry, I have measured Ki-67, ER, PR and HER2/neu positivity for a series of cases of DCIS. Results. 20 ER and/or PR positive and HER2/neu negative (50%) with average PI of 0.05, 7 ER and/or PR positive and HER2/neu positive (17.5%) with average PI of 0.14, 10 ER and/or PR negative and HER2/neu positive (25%) with average PI of 0.18, and three triple negative (7.5%) with average PI of 0.18. ER and/or PR positive and HER2/neu positive group has the highest PI (p<0.001). Further, the ER and/or PR positive and HER2/neu positive group show a linear relationship between PI and average ER/PR positivity (R=0.6). PI increases with higher grades. Conclusion. PI appears to depend upon the average fraction of positive ER/PR tumor cells, possibly with a synergistic dependence when HER2/neu is positive. If ER/PR is negative, then both HER2/neu positive and the triple negative cases appear to cluster around an average PI that is higher than the average PI in HER2/neu negative ER/PR positive negative cases. In the triple negative tumors there must be another driver of proliferation.^

Polycomb repressive complex 2 and induced basal-like breast cancer phenotype mediated by cyclin E/Cdk2

Despite of much success of breast cancer treatment, basal-like breast cancer subtype still presented as a clinical challenge to mammary oncologist for its lack of available targeted therapy owing to their negative expression of targeted molecules, such as PgR, ERα and Her2. These molecules are all critical regulators in mammary gland development. EZH2, a histone methyltransferase, by forming Polycomb Repressive Complex 2(PRC2) can directly suppress a large array of developmental regulators. Overexpression of cyclin E has also been correlated with basal-like (triple-negative) breast cancer and poor prognosis. We found an important functional link between these two molecules. Cyclin E/Cdk2 can enhance PRC2 function by phosphorylating a specific residue of EZH2, threonine 416 and increasing EZH2's ability to complex with SUZ12. This regulation would further recruit whole PRC2 complex to core promoter regions of these developmental regulators. The local enrichment of PRC2 complex would then trimethylate H3K27 around the core promoter regions and suppress the expression of targeted genes, which included PgR, ERα, erbB2 and BRCA1. This widespread gene suppressive effect imposed by highly active PRC2 complex would then transform the lumina) type cell to adopt a basal-like phenotype. This finding suggested deregulated Cdk2 activity owing to cyclin E overexpression may contribute to basal phenotype through enhancing epigenetic silencing effects by regulating PRC2 function. Inhibition of Cdk2 activity in basal-like cancer cells may help release the suppression, reexpress the silenced genes and become responsive to existing anti-hormone or anti-Her2 therapy. From this study, the mechanisms described here provided a rationale to target basal-like breast cancer by new combinational therapy of Cdk2 inhibitors together with Lapatinib, or Aromatin. ^

Breast cancer risk factors among Mexican-American women

Introduction: The average age of onset of breast cancer among Hispanic women is 50 years, more than a decade earlier than non-Hispanic white women. Age at diagnosis is an important prognostic factor for breast cancer; younger age at onset is more likely to be associated with advanced disease, poorer prognosis, hormone receptor negative breast tumors, and a greater likelihood of hereditary breast cancer. Studies of breast cancer risk factors including reproductive risk factors, family history of breast cancer, and breast cancer subtype have been conducted predominately in non-Hispanic whites. Breast cancer is a heterogeneous disease with the presence of clinically, biologically, and epidemiologically distinct subtypes that also differ with respect to their risk factors. The associations between reproductive risk factors and family history of breast cancer have been well documented in the literature. However, only a few studies have assessed these associations with breast cancer subtype in Hispanic populations. Methods: To assess the associations between reproductive risk factors and family history of breast cancer we conducted three separate studies. First, we conducted a case-control study of 172 Mexican-American breast cancer cases and 344 age matched controls residing in Harris County, TX to assess reproductive and other risk factors. We conducted logistic regression analysis to assess differences in cases and controls adjusted for age at diagnosis and birthplace and then we conducted a multinomial logistic regression analysis to compare reproductive risk factors among the breast tumor subtypes. In a second study, we identified 139 breast cancer patients with a first- or second-degree family history of breast cancer and 298 without a family history from the ELLA Bi-National Breast Cancer Study. In this analysis, we also computed a multinomial logistic regression to evaluate associations between family history of breast cancer and breast cancer subtypes, and logistic regression to estimate associations between breast cancer screening practices with family history of breast cancer. In the final study, we employed a cross-sectional study design in 7279 Mexican-American women in the Mano a Mano Cohort Study. We evaluated associations with family history of breast cancer and breast cancer risk factors including body mass index (BMI), lifestyle factors, migration history, and adherence to American Cancer Society (ACS) guidelines. Results: In the results of our first analyses, reproductive risk factors differed in the magnitude and direction of associations when stratified by age and birthplace among cases and controls. In our second study, family history of breast cancer, and having at least one relative diagnosed at an early age (<50 years) was associated with triple negative breast cancer (TNBC). Mammography prior to receiving a breast cancer diagnosis was associated with family history of breast cancer. In our third study that assessed lifestyle factors, migration history and family history of breast cancer; we found that women with a first-degree family history of breast cancer were more overweight or obese compared with their counterparts without a family history. There was no indication that having a family history contributed to women practicing healthier lifestyle behaviors and/or adhering to the ACS guidelines for cancer prevention. Conclusions: We observed that among Mexican-American women, reproductive risk factors were associated with breast cancer where the woman was born (US or Mexico). Having a family history of breast cancer, especially having either a first- or second-degree relative diagnosed at a younger age, was strongly associated with TNBC subtype. These results are consistent with other published studies in this area. Further, our results indicate that women with strong family histories of breast cancer are more likely to undertake mammography but not to engage in healthier lifestyle behaviors.^