Zhigang C. Wang

A Pleiotropically Acting MicroRNA, miR-31, Inhibits Breast Cancer Metastasis

MicroRNAs are well suited to regulate tumor metastasis because of their capacity to coordinately repress numerous target genes, thereby potentially enabling their intervention at multiple steps of the invasion-metastasis cascade. We identify a microRNA exemplifying these attributes, miR-31, whose expression correlates inversely with metastasis in human breast cancer patients. Overexpression of miR-31 in otherwise-aggressive breast tumor cells suppresses metastasis. We deploy a stable microRNA sponge strategy to inhibit miR-31 in vivo; this allows otherwise-nonaggressive breast cancer cells to metastasize. These phenotypes do not involve confounding influences on primary tumor development and are specifically attributable to miR-31-mediated inhibition of several steps of metastasis, including local invasion, extravasation or initial survival at a distant site, and metastatic colonization. Such pleiotropy is achieved via coordinate repression of a cohort of metastasis-promoting genes, including RhoA. Indeed, RhoA re-expression partially reverses miR-31-imposed metastasis suppression. These findings indicate that miR-31 uses multiple mechanisms to oppose metastasis.

Efficacy of Neoadjuvant Cisplatin in Triple-Negative Breast Cancer

PURPOSE Cisplatin is a chemotherapeutic agent not used routinely for breast cancer treatment. As a DNA cross-linking agent, cisplatin may be effective treatment for hereditary BRCA1-mutated breast cancers. Because sporadic triple-negative breast cancer (TNBC) and BRCA1-associated breast cancer share features suggesting common pathogenesis, we conducted a neoadjuvant trial of cisplatin in TNBC and explored specific biomarkers to identify predictors of response. PATIENTS AND METHODS Twenty-eight women with stage II or III breast cancers lacking estrogen and progesterone receptors and HER2/Neu (TNBC) were enrolled and treated with four cycles of cisplatin at 75 mg/m(2) every 21 days. After definitive surgery, patients received standard adjuvant chemotherapy and radiation therapy per their treating physicians. Clinical and pathologic treatment response were assessed, and pretreatment tumor samples were evaluated for selected biomarkers. Results Six (22%) of 28 patients achieved pathologic complete responses, including both patients with BRCA1 germline mutations;18 (64%) patients had a clinical complete or partial response. Fourteen (50%) patients showed good pathologic responses (Miller-Payne score of 3, 4, or 5), 10 had minor responses (Miller-Payne score of 1 or 2), and four (14%) progressed. All TNBCs clustered with reference basal-like tumors by hierarchical clustering. Factors associated with good cisplatin response include young age (P = .001), low BRCA1 mRNA expression (P = .03), BRCA1 promoter methylation (P = .04), p53 nonsense or frameshift mutations (P = .01), and a gene expression signature of E2F3 activation (P = .03). CONCLUSION Single-agent cisplatin induced response in a subset of patients with TNBC. Decreased BRCA1 expression may identify subsets of TNBCs that are cisplatin sensitive. Other biomarkers show promise in predicting cisplatin response.

Autocrine TGF-β and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-β and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-β and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

Evidence that Inositol Polyphosphate 4-Phosphatase Type II Is a Tumor Suppressor that Inhibits PI3K Signaling

We report that knocking down the expression of inositol polyphosphate 4-phosphatase type II (INPP4B) in human epithelial cells, like knockdown of PTEN, resulted in enhanced Akt activation and anchorage-independent growth and enhanced overall motility. In xenograft experiments, overexpression of INPP4B resulted in reduced tumor growth. INPP4B preferentially hydrolyzes phosphatidylinositol-3,4-bisphosphate (PI(3,4)P(2)) with no effect on phosphatidylinositol-3.4.5-triphosphate (PI(3,4,5)P(3)), suggesting that PI(3,4)P(2) and PI(3,4,5)P(3) may cooperate in Akt activation and cell transformation. Dual knockdown of INPP4B and PTEN resulted in cellular senescence. Finally, we found loss of heterozygosity (LOH) at the INPP4B locus in a majority of basal-like breast cancers, as well as in a significant fraction of ovarian cancers, which correlated with lower overall patient survival, suggesting that INPP4B is a tumor suppressor.

Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer

Adjuvant chemotherapy for breast cancer after surgery has effectively lowered metastatic recurrence rates. However, a considerable proportion of women suffer recurrent cancer at distant metastatic sites despite adjuvant treatment. Identification of the genes crucial for tumor response to specific chemotherapy drugs is a challenge but is necessary to improve outcomes. By using integrated genomics, we identified a small number of overexpressed and amplified genes from chromosome 8q22 that were associated with early disease recurrence despite anthracycline-based adjuvant chemotherapy. We confirmed the association in an analysis of multiple independent cohorts. SiRNA-mediated knockdown of either of two of these genes, the antiapoptotic gene YWHAZ and a lysosomal gene LAPTM4B, sensitized tumor cells to anthracyclines, and overexpression of either of the genes induced anthracycline resistance. Overexpression of LAPTM4B resulted in sequestration of the anthracycline doxorubicin, delaying its appearance in the nucleus. Overexpression of these two genes was associated with poor tumor response to anthracycline treatment in a neoadjuvant chemotherapy trial in women with primary breast cancer. Our results suggest that 8q22 amplification and overexpression of LAPTM4B and YWHAZ contribute to de novo chemoresistance to anthracyclines and are permissive for metastatic recurrence. Overexpression of these two genes may predict anthracycline resistance and influence selection of chemotherapy.

Loss of Heterozygosity and Its Correlation with Expression Profiles in Subclasses of Invasive Breast Cancers

Gene expression array profiles identify subclasses of breast cancers with different clinical outcomes and different molecular features. The present study attempted to correlate genomic alterations (loss of heterozygosity; LOH) with subclasses of breast cancers having distinct gene expression signatures. Hierarchical clustering of expression array data from 89 invasive breast cancers identified four major expression subclasses. Thirty-four of these cases representative of the four subclasses were microdissected and allelotyped using genome-wide single nucleotide polymorphism detection arrays (Affymetrix, Inc.). LOH was determined by comparing tumor and normal single nucleotide polymorphism allelotypes. A newly developed statistical tool was used to determine the chromosomal regions of frequent LOH. We found that breast cancers were highly heterogeneous, with the proportion of LOH ranging widely from 0.3% to >60% of heterozygous markers. The most common sites of LOH were on 17p, 17q, 16q, 11q, and 14q, sites reported in previous LOH studies. Signature LOH events were discovered in certain expression subclasses. Unique regions of LOH on 5q and 4p marked a subclass of breast cancers with “basal-like” expression profiles, distinct from other subclasses. LOH on 1p and 16q occurred preferentially in a subclass of estrogen receptor-positive breast cancers. Finding unique LOH patterns in different groups of breast cancer, in part defined by expression signatures, adds confidence to newer schemes of molecular classification. Furthermore, exclusive association between biological subclasses and restricted LOH events provides rationale to search for targeted genes.

A role for the scaffolding adapter GAB2 in breast cancer

The scaffolding adapter GAB2 maps to a region (11q13-14) commonly amplified in human breast cancer, and is overexpressed in breast cancer cell lines and primary tumors, but its functional role in mammary carcinogenesis has remained unexplored. We found that overexpression of GAB2 (Grb2-associated binding protein 2) increases proliferation of MCF10A mammary cells in three-dimensional culture. Coexpression of GAB2 with antiapoptotic oncogenes causes lumenal filling, whereas coexpression with Neu (also known as ErbB2 and HER2) results in an invasive phenotype. These effects of GAB2 are mediated by hyperactivation of the Shp2-Erk pathway. Furthermore, overexpression of Gab2 potentiates, whereas deficiency of Gab2 ameliorates, Neu-evoked breast carcinogenesis in mice. Finally, GAB2 is amplified in some GAB2-overexpressing human breast tumors. Our data suggest that GAB2 may be a key gene within an 11q13 amplicon in human breast cancer and propose a role for overexpression of GAB2 in mammary carcinogenesis. Agents that target GAB2 or GAB2-dependent pathways may be useful for treating breast tumors that overexpress GAB2 or HER2 or both.

Predicting features of breast cancer with gene expression patterns

Data from gene expression arrays hold an enormous amount of biological information. We sought to determine if global gene expression in primary breast cancers contained information about biologic, histologic, and anatomic features of the disease in individual patients. Microarray data from the tumors of 129 patients were analyzed for the ability to predict biomarkers [estrogen receptor (ER) and HER2], histologic features [grade and lymphatic-vascular invasion (LVI)], and stage parameters (tumor size and lymph node metastasis). Multiple statistical predictors were used and the prediction accuracy was determined by cross-validation error rate; multidimensional scaling (MDS) allowed visualization of the predicted states under study. Models built from gene expression data accurately predict ER and HER2 status, and divide tumor grade into high-grade and low-grade clusters; intermediate-grade tumors are not a unique group. In contrast, gene expression data is inaccurate at predicting tumor size, lymph node status or LVI. The best model for prediction of nodal status included tumor size, LVI status and pathologically defined tumor subtype (based on combinations of ER, HER2, and grade); the addition of microarray-based prediction to this model failed to improve the prediction accuracy. Global gene expression supports a binary division of ER, HER2, and grade, clearly separating tumors into two categories; intermediate values for these bio-indicators do not define intermediate tumor subsets. Results are consistent with a model of regional metastasis that depends on inherent biologic differences in metastatic propensity between breast cancer subtypes, upon which time and chance then operate.

BRCA1 promoter methylation in sporadic breast tumors: relationship to gene expression profiles.

BRCA1 is a tumor suppressor gene that functions in DNA repair. Basal-like tumors are a distinctive subtype of breast cancer defined by gene expression profiles. Hereditary BRCA1 breast tumors and basal-like sporadic tumors have a similar phenotype and gene expression signature, suggesting involvement of BRCA1 in the pathogenesis of sporadic basal-like cancer. This study evaluates the role of BRCA1 in sporadic breast tumorigenesis. BRCA1 protein expression and promoter methylation are compared to tumor histopathology and gene expression profiles. We find BRCA1 protein expression correlates with tumor mitotic rate, consistent with normal cell-cycle regulation of the BRCA1 gene. Methylation is found in 21% of tumors and is associated with lower BRCA1 protein, but not with specific pathologic features. Basal-like tumors, defined by hierarchical clustering of gene expression, have infrequent BRCA1 methylation and high levels of BRCA1 protein expression consistent with their high mitotic rate. Tumors with BRCA1 promoter methylation are present in all expression clusters; however, a subgroup of ER-positive high-grade tumors has a significantly greater number of BRCA1 methylated tumors. Absence of BRCA1 promoter methylation and high levels of BRCA1 expression in basal-like sporadic tumors suggest alternate explanations for the phenotypic similarities of these tumors to hereditary BRCA1 tumors.

Telomeric Allelic Imbalance Indicates Defective DNA Repair and Sensitivity to DNA-Damaging Agents

UNLABELLED DNA repair competency is one determinant of sensitivity to certain chemotherapy drugs, such as cisplatin. Cancer cells with intact DNA repair can avoid the accumulation of genome damage during growth and also can repair platinum-induced DNA damage. We sought genomic signatures indicative of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (N(tAI)) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of N(tAI) forecast a better initial response. We found an inverse relationship between BRCA1 expression and N(tAI) in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation. Thus, accumulation of telomeric allelic imbalance is a marker of platinum sensitivity and suggests impaired DNA repair. SIGNIFICANCE Mutations in BRCA genes cause defects in DNA repair that predict sensitivity to DNA damaging agents, including platinum; however, some patients without BRCA mutations also benefit from these agents. NtAI, a genomic measure of unfaithfully repaired DNA, may identify cancer patients likely to benefit from treatments targeting defective DNA repair.