Genes responsive to both oxidant stress and loss of estrogen receptor function identify a poor prognosis group of estrogen receptor positive primary breast cancers

Buck Institute for Age Research, Redwood Boulevard, Novato, California 94945, USA.
Breast cancer research: BCR (Impact Factor: 5.88). 07/2008; 10(4):R61. DOI: 10.1186/bcr2120
Source: PubMed

ABSTRACT Oxidative stress can modify estrogen receptor (ER) structure and function, including induction of progesterone receptor (PR), altering the biology and clinical behavior of endocrine responsive (ER-positive) breast cancer.
To investigate the impact of oxidative stress on estrogen/ER-regulated gene expression, RNA was extracted from ER-positive/PR-positive MCF7 breast cancer cells after 72 hours of estrogen deprivation, small-interfering RNA knockdown of ER-alpha, short-term (8 hours) exposure to various oxidant stresses (diamide, hydrogen peroxide, and menadione), or simultaneous ER-alpha knockdown and oxidant stress. RNA samples were analyzed by high-throughput expression microarray (Affymetrix), and significance analysis of microarrays was used to define gene signatures responsive to estrogen/ER regulation and oxidative stress. To explore the association of these signatures with breast cancer biology, microarray data were analyzed from 394 ER-positive primary human breast cancers pooled from three independent studies. In particular, an oxidant-sensitive estrogen/ER-responsive gene signature (Ox-E/ER) was correlated with breast cancer clinical parameters and disease-specific patient survival (DSS).
From 891 estrogen/ER-regulated probes, a core set of 75 probes (62 unique genes) responsive to all three oxidants were selected (Ox-E/ER signature). Ingenuity pathway analysis of this signature highlighted networks involved in development, cancer, and cell motility, with intersecting nodes at growth factors (platelet-derived growth factor-BB, transforming growth factor-beta), a proinflammatory cytokine (tumor necrosis factor), and matrix metalloproteinase-2. Evaluation of the 394 ER-positive primary breast cancers demonstrated that Ox-E/ER index values correlated negatively with PR mRNA levels (rp = -0.2; P = 0.00011) and positively with tumor grade (rp = 0.2; P = 9.741 x e-5), and were significantly higher in ER-positive/PR-negative versus ER-positive/PR-positive breast cancers (t-test, P = 0.0008). Regardless of PR status, the Ox-E/ER index associated with reduced DSS (n = 201; univariate Cox, P = 0.078) and, using the optimized cut-point, separated ER-positive cases into two significantly different DSS groups (log rank, P = 0.0009).
An oxidant-sensitive subset of estrogen/ER-responsive breast cancer genes linked to cell growth and invasion pathways was identified and associated with loss of PR and earlier disease-specific mortality, suggesting that oxidative stress contributes to the development of an aggressive subset of primary ER-positive breast cancers.

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    ABSTRACT: Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer related deaths among Canadian women. Development of distant metastases is the leading cause of morbidity and mortality from this disease. Breast cancer is a highly heterogeneous disease that is amenable to intervention with targeted therapeutics; however, therapies that are currently available have limited efficacy in the metastatic setting. To identify novel molecular mediators of breast cancer bone metastasis that might also serve as therapeutic targets, we subjected 4T1 mammary carcinoma cells to in vivo selection in Balb/c mice and isolated sub-populations with an aggressively bone-metastatic phenotype. Gene expression profiling of these cells revealed Glycoprotein NMB (GPNMB), also known as Osteoactivin, as a gene that was highly expressed in bone metastatic breast cancer cells. GPNMB is a type I transmembrane, cell surface expressed protein with an extracellular RGD and PKD domains and a cytoplasmic hemITAM signaling motif that had not previously been implicated in breast cancer. We demonstrate that ectopic GPNMB expression was sufficient to promote migration and invasion of breast cancer cells in vitro and the formation of bone metastases in vivo.Subsequently, we analyzed GPNMB mRNA and protein expression levels in hundreds of breast tumors and found that GPNMB expression positively correlates with increased risk of metastasis and shorter overall survival times. We have also demonstrated that GPNMB is most commonly expressed in breast tumors belonging to the triple negative subtype, for which there are no targeted therapies currently available. We showed for the first time that CDX-011, a GPNMB-targeted monoclonal antibody-drug conjugate, was capable of killing GPNMB-expressing breast cancer cells in vitro and inducing tumor regression in vivo. Finally, we investigated the effects of GPNMB on primary tumor progression and found that it inhibits tumor cell apoptosis while enhancing angiogenesis and tumor growth in vivo. We demonstrate that the extracellular domain (ECD) of GPNMB can be proteolytically cleaved and shed from the surface of breast cancer cells, which is mediated by ADAM10. We postulated that the shed extracellular domain (ECD) of GPNMB might be responsible for some of its pro-angiogenic effects and showed that this ECD was indeed capable of inducing endothelial cell migration in vitro.The body of work described in this thesis is the first to identify GPNMB as a functional mediator of breast cancer growth and metastasis and to validate it as an important clinical target in human breast cancer. Le cancer du sein est le cancer le plus fréquemment diagnostiqué et la seconde cause de mortalité associée au cancer chez les femmes canadiennes. Le développement de métastases est la cause majeure de la morbidité et de la mortalité dûes à cette maladie. Le cancer du sein est une maladie très hétérogène qui peut toutefois être traité par l'utilisation de thérapie ciblée ; toutefois, les thérapies actuellement disponibles ont un effet limité sur la formation des métastases. Dans le but d'identifier de nouveaux médiateurs moléculaires associés à la formation de métastases osseuses dérivées du cancer du sein et qui pourraient être utilisés comme cibles thérapeutiques, nous avons soumis les cellules de carcinome mammaire 4T1 à un processus de sélection in vivo dans des souris Balb/c. Nous avons ainsi isolé des sous-populations de cellules caractérisées par leur agressivité à former des métastases osseuses. L'étude de l'expression génique de ces cellules a mis en évidence que le gène codant pour la Glycoprotéine NMB (GPNMB), aussi connu sous le nom de Ostéoactivine, est très fortement exprimé dans les lignées de cancer du sein métastatiques pour l'os.GPNMB est une protéine de surface transmembranaire de type I qui possède des domaines RGD et PKD extracellulaires ainsi qu'un motif hemITAM de signalisation cytoplasmique et n'avait encore jamais été rapportée comme impliquée dans le cancer du sein.Nous avons démontré que l'expression ectopique de GPNMB était suffisante pour promouvoir la migration et l'invasion de cellules de cancer du sein in vitro ainsi que la formation de métastases in vivo.Par la suite, nous avons analysé les niveaux d'expression des ARNm et de la protéine GPNMB dans des centaines de tumeur du sein humain et avons observé que l'expression de GPNMB corrèle positivement avec un risque accru de présence de métastases ainsi qu'une réduction du temps moyen de survie. Nous avons également démontré que GPNMB est le plus fréquemment exprimé dans des tumeurs mammaires appartenant au sous-type triple négatif pour lequel il n'y a actuellement aucune thérapie ciblée disponible.Par ailleurs, nous montrons pour la première fois que CDX-011, une drogue conjuguée à un anticorps monoclonal reconnaissant GPNMB, était capable, in vitro, d'éradiquer spécifiquement les cellules de cancer du sein exprimant GPNMB ainsi que d'induire une régression tumorale in vivo.Finalement, nous avons déterminé les effets de GPNMB sur la progression des tumeurs primaires et avons observé que GPNMB inhibait l'apoptose des cellules tumorales tout en augmentant l'angiogenèse et la croissance tumorale in vivo. Nous avons démontré que le domaine extracellulaire de GPNMB (ECD) pouvait être clivé de façon protéolytique par ADAM10 et ainsi être libéré de la surface cellulaire des cellules de cancer du sein. 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