Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, - 2.2% to 6.8%; P

BRCA1 and GATA3 corepress FOXC1 to inhibit the pathogenesis of basal-like breast cancers

In this study we describe a novel interaction between the breast/ovarian tumor suppressor gene BRCA1 and the transcription factor GATA3, an interaction, which is important for normal breast differentiation. We show that the BRCA1-GATA3 interaction is important for the repression of genes associated with triple-negative and basal-like breast cancer (BLBCs) including FOXC1, and that GATA3 interacts with a C-terminal region of BRCA1. We demonstrate that FOXC1 is an essential survival factor maintaining the proliferation of BLBCs cell lines. We define the mechanistic basis of this corepression and identify the GATA3-binding site within the FOXC1 distal promoter region. We show that BRCA1 and GATA3 interact on the FOXC1 promoter and that BRCA1 requires GATA3 for recruitment to this region. This interaction requires fully functional BRCA1 as a mutant BRCA1 protein is unable to localize to the FOXC1 promoter or repress FOXC1 expression. We demonstrate that this BRCA1-GATA3 repression complex is not a FOXC1-specific phenomenon as a number of other genes associated with BLBCs such as FOXC2, CXCL1 and p-cadherin were also repressed in a similar manner. Finally, we demonstrate the importance of our findings by showing that loss of GATA3 expression or aberrant FOXC1 expression contributes to the drug resistance and epithelial-to-mesenchymal transition-like phenotypes associated with aggressive BLBCs. Oncogene (2012) 31, 3667-3678; doi:10.1038/onc.2011.531; published online 28 November 2011

JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer.

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P <0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

Background: There is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.

Methods: DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided.

Results: In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population.

Conclusions: A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

TBX2 represses CST6 resulting in uncontrolled legumain activity to sustain breast cancer proliferation: a novel cancer-selective target pathway with therapeutic opportunities.

TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.

Local recurrence in patients with large and locally advanced breast cancer treated with primary chemotherapy

Background: Primary chemotherapy is being given in the treatment of large and locally advanced breast cancers, but a major concern is local relapse after therapy. This paper has examined patients treated with primary chemotherapy and surgery (either breast-conserving surgery or mastectomy) and has examined the role of factors which may indicate those patients who are subsequently more likely to experience local recurrence of,disease.

Methods: A consecutive series of 173 women, with data available for 166 of these, presenting with large and locally advanced breast cancer (T2 >4 cm, T3, T4, or N2) were treated with primary chemotherapy comprising cyclophosphamide, vincristine, doxorubicin, and prednisolone and then surgery (either conservation or mastectomy with axillary surgery) followed by radiotherapy were examined.

Results: The clinical response rate of these patients was 75% (21% complete and 54% partial), with a complete pathological response rate of 15%. A total of 10 patients (6%) experienced local disease relapse, and the median time to relapse was 14 months (ranging from 3 to 40). The median survival in this group was 27 months (ranging from 13 to 78). In patients having breast conservation surgery, local recurrence occurred in 2%, and in those undergoing mastectomy 7% experience local relapse of disease. Factors predicting patients most likely to experience local recurrence were poor clinical response and residual axillary nodal disease after chemotherapy.

Conclusions: Excellent local control of disease can be achieved in patients with large and locally advanced breast cancers using a combination of primary chemotherapy, surgery and radiotherapy. However, the presence of residual tumor in the axillary lymph nodes after chemotherapy is a predictor of local recurrence and patients with a better clinical response were also less likely to experience local disease recurrence. The size and degree of pathological response did not predict patients most likely to experience recurrence of disease. (C) 2003 Excerpta Medica, Inc. All rights reserved.

Immunoselection of breast and ovarian cancer cells with trastuzumab and natural killer cells:selective escape of CD44high/CD24low/HER2low breast cancer stem cells

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44(high)CD24(low) "cancer stem cell-like" phenotype and expressed significantly less HER2 compared with non-stem cells. Likewise, the CD44(high)CD24(low) population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44(high)CD24(low) cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants.

The prognostic significance of the aberrant extremes of p53 immunophenotypes in breast cancer

Aims: The utility of p53 as a prognostic assay has been elusive. The aims of this study were to describe a novel, reproducible scoring system and assess the relationship between differential p53 immunohistochemistry (IHC) expression patterns, TP53 mutation status and patient outcomes in breast cancer.

Methods and Results: Tissue microarrays were used to study p53 IHC expression patterns: expression was defined as extreme positive (EP), extreme negative (EN), and non-extreme (NE; intermediate patterns). Overall survival (OS) was used to define patient outcome. A representative subgroup (n = 30) showing the various p53 immunophenotypes was analysed for TP53 hotspot mutation status (exons 4-9). Extreme expression of any type occurred in 176 of 288 (61%) cases. As compared with NE expression, EP expression was significantly associated (P = 0.039) with poorer OS. In addition, as compared with NE expression, EN expression was associated (P = 0.059) with poorer OS. Combining cases showing either EP or EN expression better predicted OS than either pattern alone (P = 0.028). This combination immunophenotype was significant in univariate but not multivariate analysis. In subgroup analysis, six substitution exon mutations were detected, all corresponding to extreme IHC phenotypes. Five missense mutations corresponded to EP staining, and the nonsense mutation corresponded to EN staining. No mutations were detected in the NE group.

Conclusions: Patients with extreme p53 IHC expression have a worse OS than those with NE expression. Accounting for EN as well as EP expression improves the prognostic impact. Extreme expression positively correlates with nodal stage and histological grade, and negatively with hormone receptor status. Extreme expression may relate to specific mutational status.

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

Manganese Superoxide Dismutase Is a Promising Target for Enhancing Chemosensitivity of Basal-Like Breast Carcinoma

AIMS: Although earlier reports highlighted a tumor suppressor role for manganese superoxide dismutase (MnSOD), recent evidence indicates increased expression in a variety of human cancers including aggressive breast carcinoma. In the present article, we hypothesized that MnSOD expression is significantly amplified in the aggressive breast carcinoma basal subtype, and targeting MnSOD could be an attractive strategy for enhancing chemosensitivity of this highly aggressive breast cancer subtype.

RESULTS: Using MDA-MB-231 and BT549 as a model of basal breast cancer cell lines, we show that knockdown of MnSOD decreased the colony-forming ability and sensitized the cells to drug-induced cell death, while drug resistance was associated with increased MnSOD expression. In an attempt to develop a clinically relevant approach to down-regulate MnSOD expression in patients with basal breast carcinoma, we employed activation of the peroxisome proliferator-activated receptor gamma (PPARγ) to repress MnSOD expression; PPARγ activation significantly reduced MnSOD expression, increased chemosensitivity, and inhibited tumor growth. Moreover, as a proof of concept for the clinical use of PPARγ agonists to decrease MnSOD expression, biopsies derived from breast cancer patients who had received synthetic PPARγ ligands as anti-diabetic therapy had significantly reduced MnSOD expression. Finally, we provide evidence to implicate peroxynitrite as the mechanism involved in the increased sensitivity to chemotherapy induced by MnSOD repression.

INNOVATION AND CONCLUSION: These data provide evidence to link increased MnSOD expression with the aggressive basal breast cancer, and underscore the judicious use of PPARγ ligands for specifically down-regulating MnSOD to increase the chemosensitivity of this subtype of breast carcinoma.

CD44 increases the efficiency of distant metastasis of breast cancer

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients.

FKBPL: a marker of good prognosis in breast cancer

FK506-binding protein-like (FKBPL) has established roles as an anti-tumor protein, with a therapeutic peptide based on this protein, ALM201, shortly entering phase I/II clinical trials. Here, we evaluated FKBPL's prognostic ability in primary breast cancer tissue, represented on tissue microarrays (TMA) from 3277 women recruited into five independent retrospective studies, using immunohistochemistry (IHC). In a meta-analysis, FKBPL levels were a significant predictor of BCSS; low FKBPL levels indicated poorer breast cancer specific survival (BCSS) (hazard ratio (HR) = 1.30, 95% confidence interval (CI) 1.14-1.49, p < 0.001). The prognostic impact of FKBPL remained significant after adjusting for other known prognostic factors (HR = 1.25, 95% CI 1.07-1.45, p = 0.004). For the sub-groups of 2365 estrogen receptor (ER) positive patients and 1649 tamoxifen treated patients, FKBPL was significantly associated with BCSS (HR = 1.34, 95% CI 1.13-1.58, p < 0.001, and HR = 1.25, 95% CI 1.04-1.49, p = 0.02, respectively). A univariate analysis revealed that FKBPL was also a significant predictor of relapse free interval (RFI) within the ER positive patient group, but it was only borderline significant within the smaller tamoxifen treated patient group (HR = 1.32 95% CI 1.05-1.65, p = 0.02 and HR = 1.23 95% CI 0.99-1.54, p = 0.06, respectively). The data suggests a role for FKBPL as a prognostic factor for BCSS, with the potential to be routinely evaluated within the clinic.

Protein deregulation associated with breast cancer metastasis

Breast cancer is one of the most prevalent malignancies worldwide. It consists of a group of tumor cells that have the ability to grow uncontrollably, overcome replicative senescence (tumor progression) and metastasize within the body. Metastases are processes that consist of an array of complex gene dysregulation events. Although these processes are still not fully understood, the dysregulation of a number of key proteins must take place if the tumor cells are to disseminate and metastasize. It is now widely accepted that future effective and innovative treatments of cancer metastasis will have to encompass all the major components of malignant transformation. For this reason, much research is now being carried out into the mechanisms that govern the malignant transformation processes. Recent research has identified key genes involved in the development of metastases, as well as their mechanisms of action. A detailed understanding of the encoded proteins and their interrelationship generates the possibility of developing novel therapeutic approaches. This review will focus on a select group of proteins, often deregulated in breast cancer metastasis, which have shown therapeutic promise, notably, EMT, E-cadherin, Osteopontin, PEA3, Transforming Growth Factor Beta (TGF-β) and Ran.