Alejandro Contreras

Comprehensive Genomic Analysis Identifies Novel Subtypes and Targets of Triple-Negative Breast Cancer

Purpose: Genomic profiling studies suggest that triple-negative breast cancer (TNBC) is a heterogeneous disease. In this study, we sought to define TNBC subtypes and identify subtype-specific markers and targets. Experimental Design: RNA and DNA profiling analyses were conducted on 198 TNBC tumors [estrogen receptor (ER) negativity defined as Allred scale value ≤ 2] with >50% cellularity (discovery set: n = 84; validation set: n = 114) collected at Baylor College of Medicine (Houston, TX). An external dataset of seven publically accessible TNBC studies was used to confirm results. DNA copy number, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed independently using these datasets. Results: We identified and confirmed four distinct TNBC subtypes: (i) luminal androgen receptor (AR; LAR), (ii) mesenchymal (MES), (iii) basal-like immunosuppressed (BLIS), and (iv) basal-like immune-activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (log-rank test: P = 0.042 and 0.041, respectively) and DSS (log-rank test: P = 0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA) and suggested that gene amplification drives gene expression in some cases [FGFR2 (BLIS)]. Putative subtype-specific targets were identified: (i) LAR: androgen receptor and the cell surface mucin MUC1, (ii) MES: growth factor receptors [platelet-derived growth factor (PDGF) receptor A; c-Kit], (iii) BLIS: an immunosuppressing molecule (VTCN1), and (iv) BLIA: Stat signal transduction molecules and cytokines. Conclusion: There are four stable TNBC subtypes characterized by the expression of distinct molecular profiles that have distinct prognoses. These studies identify novel subtype-specific targets that can be targeted in the future for the effective treatment of TNBCs. Clin Cancer Res; 21(7); 1688–98. ©2014 AACR. See related commentary by Vidula and Rugo, p. 1511

Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland

To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus–long terminal repeat–driven, conditional, fibroblast growth factor (FGF)–independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in iFGFR1 tyrosine phosphorylation, increased proliferation, activation of mitogen-activated protein kinase and Akt, and lateral budding. Lateral buds appeared as early as 3 d after AP20187 treatment and initially consisted of bilayered epithelial cells and displayed apical and basolateral polarity appeared after 13 d of AP20187 treatment. Invasive lesions characterized by multicell-layered lateral buds, decreased myoepithelium, increased vascular branching, and loss of cell polarity were observed after 2–4 wk of treatment. These data indicate that acute iFGFR1 signaling results in increased lateral budding of the mammary ductal epithelium, and that sustained activation induces alveolar hyperplasia and invasive lesions.

The dynamic mobility of histone H1 is regulated by cyclin/CDK phosphorylation

ABSTRACT The linker histone H1 is involved in maintaining higher-order chromatin structures and displays dynamic nuclear mobility, which may be regulated by posttranslational modifications. To analyze the effect of H1 tail phosphorylation on the modulation of the histones nuclear dynamics, we generated a mutant histone H1, referred to as M1-5, in which the five cyclin-dependent kinase phosphorylation consensus sites were mutated from serine or threonine residues into alanines. Cyclin E/CDK2 or cyclin A/CDK2 cannot phosphorylate the mutant in vitro. Using the technique of fluorescence recovery after photobleaching, we observed that the mobility of a green fluorescent protein (GFP)-M1-5 fusion protein is decreased compared to that of a GFP-wild-type H1 fusion protein. In addition, recovery of H1 correlated with CDK2 activity, as GFP-H1 mobility was decreased in cells with low CDK2 activity. Blocking the activity of CDK2 by p21 expression decreased the mobility of GFP-H1 but not that of GFP-M1-5. Finally, the level and rate of recovery of cyan fluorescent protein (CFP)-M1-5 were lower than those of CFP-H1 specifically in heterochromatic regions. These data suggest that CDK2 phosphorylates histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions.

Thermal Enhancement with Optically Activated Gold Nanoshells Sensitizes Breast Cancer Stem Cells to Radiation Therapy

Local hyperthermia with gold nanoshells sensitizes cancer stem cells to radiation treatment in mouse and human breast cancer preclinical models. A Midas Touch for Breast Cancer Treatment One of the biggest hurdles to beating breast cancer is that a small population of stem cell–like cells (CSCs) within the tumor are stubbornly resistant to radiation therapy and chemotherapy. It is this CSC subpopulation that is responsible for relapse after successful treatment with radiation and drugs. Atkinson and colleagues now take a nanotech approach to solving this problem. Working in two different mouse models of breast cancer, they use gold nanoshells to turn up the heat on CSCs, making them more sensitive to radiation therapy. To sensitize breast tumor CSCs to radiation treatment, Atkinson and colleagues engineered gold nanoshells (nanoparticles comprising a silica core with an ultrathin gold layer) that accumulate preferentially in solid tumors and can be activated by near-infrared light (which is able to penetrate tissues). When activated by a laser, the nanoshells cause local heating of the tumors in which they have accumulated. The investigators first tested their gold nanoshells in mice bearing breast tumors that were particularly aggressive and radioresistant. Using surface markers and flow cytometry, the authors found that these breast tumors contained a cell population similar to the CSCs of human breast tumors. They injected their gold nanoshells intravenously into the tumor-bearing mice and then exposed the animals to both near-infrared laser light and 6 gray of ionizing radiation. This dual treatment not only shrank the tumors but also decreased the number of CSCs. Atkinson and colleagues then transplanted the treated tumors into syngeneic recipient mice and found that the tumors had become less aggressive and more differentiated in response to the dual laser radiation treatment. The researchers then went a step further, repeating these experiments with human breast tumor biopsy samples propagated in mice. Once again, they saw that the nanoshell-induced heating effect rendered the human breast tumors and their CSCs much more sensitive to ionizing radiation. But how does the combined treatment work? The investigators demonstrated that nanoshell-induced heating prevented breast tumor cells from repairing DNA double-strand breaks induced by ionizing radiation, resulting in an increase in their radiation sensitivity. Although the gold nanoshells still require further testing, hyperthermia treatments are already in clinical trials, and ionizing radiation is a staple of cancer therapy. This suggests that the dual hyperthermia-radiation cancer therapy of Atkinson et al. should be amenable to translation to a clinical setting. Breast cancer metastasis and disease recurrence are hypothesized to result from residual cancer stem cells, also referred to as tumor-initiating cells, which evade initial treatment. Using both syngeneic mouse and human xenograft models of triple-negative breast cancer, we have demonstrated that a subpopulation enriched in cancer stem cells was more resistant to treatment with 6 gray of ionizing radiation than the bulk of the tumor cells, and accordingly their relative proportion increased 48 to 72 hours after ionizing radiation treatment. In contrast, we achieved a larger reduction in tumor size without a concomitant increase in the percentage of cancer stem cells by treating with local hyperthermia for 20 minutes at 42°C after ionizing radiation using intravenously administered, optically activated gold nanoshells. Forty-eight hours after treatment, cells derived from the tumors treated with ionizing radiation plus hyperthermia exhibited both a marked decrease in tumorigenicity and a more differentiated phenotype than mock- and ionizing radiation–treated tumors. Thus, we have confirmed that these cancer stem cells are responsible for accelerated repopulation in vivo and demonstrated that hyperthermia sensitizes this cell population to radiation treatment. These findings suggest that local hyperthermia delivered by gold nanoshells plus radiation can eliminate radioresistant breast cancer stem cells.

Tracking of multimodal therapeutic nanocomplexes targeting breast cancer in vivo.

Nanoparticle-based therapeutics with local delivery and external electromagnetic field modulation holds extraordinary promise for soft-tissue cancers such as breast cancer; however, knowledge of the distribution and fate of nanoparticles in vivo is crucial for clinical translation. Here we demonstrate that multiple diagnostic capabilities can be introduced in photothermal therapeutic nanocomplexes by simultaneously enhancing both near-infrared fluorescence and magnetic resonance imaging (MRI). We track nanocomplexes in vivo, examining the influence of HER2 antibody targeting on nanocomplex distribution over 72 h. This approach provides valuable, detailed information regarding the distribution and fate of complex nanoparticles designed for specific diagnostic and therapeutic functions.

Maspin is physically associated with β1 integrin regulating cell adhesion in mammary epithelial cells

Maspin is a tumor‐suppressor serpin (serine protease inhibitor), which inhibits cell invasion and migration. Here, we analyzed maspin function in cell adhesion in nontransformed mammary epithelial cells and investigated the underlying mechanism involved in this process. We report that maspin acts in the early steps in the cell adhesion process. Addition of recombinant maspin rapidly increased MCF‐10A cell adhesion to the endogenously deposited matrix, and conversely both an antimaspin antibody (Ab) and maspin knockdown by RNA interference resulted in decreased cell adhesion. Mutation analyses revealed that a region of 86 amino acids located between aa 139 and aa 225 was responsible for maspin effect on adhesion. In addition, we show that maspin is associated with detergent‐insoluble cortical cytoskeleton elements. Collectively, these results suggest that maspin is part of the supramolecular structure of the adhesion plaque and it modulates cell adhesion via a β1 integrin‐dependent mechanism.—Cella, N., Contreras, A., Latha, K., Rosen, J. M., and Zhang, M. Maspin is physically and functionally associated with β1 integrin regulating cell adhesion in mammary epithelial cells. FASEB J. 20, E721–E731 (2006)

Recurrent ESR1 – CCDC170 rearrangements in an aggressive subset of oestrogen receptor-positive breast cancers

Characterizing the genetic alterations leading to the more aggressive forms of estrogen receptor positive (ER+) breast cancers are of critical significance in breast cancer management. Here we identify recurrent rearrangements between estrogen receptor gene ESR1 and its neighbor CCDC170, which are enriched in the more aggressive and endocrine-resistant luminal-B tumors, through large-scale analyses of breast cancer transcriptome and copy number alterations. Further screening of 200 ER+ breast cancers identifies eight ESR1-CCDC170 positive tumors. These fusions encode N-terminally truncated CCDC170 proteins (ΔCCDC170). When introduced into ER+ breast cancer cells, ΔCCDC170 leads to markedly increased cell motility and anchorage-independent growth, reduced endocrine sensitivity, and enhanced xenograft tumor formation. Mechanistic studies suggest that ΔCCDC170 engages Gab1 signalosome to potentiate growth factor signaling and enhance cell motility. Together, this study identifies neoplastic ESR1-CCDC170 fusions in a more aggressive subset of ER+ breast cancer, which suggests a new concept of ER pathobiology in breast cancer.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase

IntroductionActivation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.MethodsAltered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.ResultsModerate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.ConclusionsModerate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

Upregulation of ER signaling as an adaptive mechanism of cell survival in HER2-positive breast tumors treated with anti-HER2 therapy

Purpose: To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples. Experimental design: Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 posttreatment) were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and Western blot analysis. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant, respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro. Results: Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy compared with untreated tumors in both preclinical models (UACC812: ER P = 0.0014; Bcl2 P < 0.001 and MCF7/HER2-18: ER P = 0.0007; Bcl2 P = 0.0306). In the neoadjuvant clinical study, lapatinib treatment for 2 weeks was associated with parallel upregulation of ER and Bcl2 (Spearman coefficient: 0.70; P = 0.0002). Importantly, 18% of tumors originally ER-negative (ER−) converted to ER+ upon anti-HER2 therapy. In ER−/HER2+ MCF7/HER2-18 xenografts, ER reexpression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (P = 0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti–HER2-resistant growth (P < 0.0001). Conclusions: HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance. Clin Cancer Res; 21(17); 3995–4003. ©2015 AACR.

Circulating and disseminated tumor cells from breast cancer patient-derived xenograft-bearing mice as a novel model to study metastasis.

IntroductionReal-time monitoring of biologic changes in tumors may be possible by investigating the transitional cells such as circulating tumor cells (CTCs) and disseminated tumor cells in bone marrow (BM-DTCs). However, the small numbers of CTCs and the limited access to bone marrow aspirates in cancer patients pose major hurdles. The goal of this study was to determine whether breast cancer (BC) patient-derived xenograft (PDX) mice could provide a constant and renewable source of CTCs and BM-DTCs, thereby representing a unique system for the study of metastatic processes.MethodsCTCs and BM-DTCs, isolated from BC PDX-bearing mice, were identified by immunostaining for human pan-cytokeratin and nuclear counterstaining of red blood cell-lysed blood and bone marrow fractions, respectively. The rate of lung metastases (LM) was previously reported in these lines. Associations between the presence of CTCs, BM-DTCs, and LM were assessed by the Fisher’s Exact and Cochran-Mantel-Haenszel tests. Two separate genetic signatures associated with the presence of CTC clusters and with lung metastatic potential were computed by using the expression arrays of primary tumors from different PDX lines and subsequently overlapped to identify common genes.ResultsIn total, 18 BC PDX lines were evaluated. CTCs and BM-DTCs, present as either single cells or clusters, were detected in 83% (15 of 18) and 62.5% (10 to16) of the lines, respectively. A positive association was noted between the presence of CTCs and BM-DTCs within the same mice. LM was previously found in 9 of 18 (50%) lines, of which all nine had detectable CTCs. The presence of LM was strongly associated with the detection of CTC clusters but not with individual cells or detection of BM-DTCs. Overlapping of the two genetic signatures of the primary PDX tumors associated with the presence of CTC clusters and with lung metastatic potential identified four genes (HLA-DP1A, GJA1, PEG3, and XIST). This four-gene profile predicted distant metastases-free survival in publicly available datasets of early BC patients.ConclusionThis study suggests that CTCs and BM-DTCs detected in BC PDX-bearing mice may represent a valuable and unique preclinical model for investigating the role of these rare cells in tumor metastases.