Estrogen controls the survival of BRCA1-deficient cells via a PI3K-NRF2-regulated pathway.

Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1's tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase-AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Retinoblastoma pathway deregulatory mechanisms determine clinical outcome in high-grade serous ovarian carcinoma.

Alterations in the retinoblastoma pathway are frequent in ovarian/tubal high-grade serous cancers, but the mechanism of deregulation and the impact on patient outcome are poorly understood. A cohort of 334 high-grade serous carcinomas was studied by immunohistochemical analysis of RB1, p16, cyclin D1, cyclin E1, and Ki67. Additional detailed analyses including RB1 allelic deletion (n=42), mutation (n=75), methylation (n=31), and SNP array analyses (n=75) were performed on cases with clinical parameters, including age, debulking status, treatment, and clinical outcome. p16/RB1 expression results yielded three distinct clinically relevant subgroups upon multivariable analysis controlling for stage, debulking status, and treatment types: p16 homogeneous/RB1+ with the shortest progression-free survival (median 15 months (95% CI: 13-18); P=0.016) compared with the p16 heterogeneous/RB1+ subgroup (median 22 months (95% CI: 16-32)) and the p16 homogeneous/RB1- subgroup (median 20 months (95% CI: 15-24)). Patients in the p16 homo/RB1- subgroup showed a significant increase in overall survival (>60 months; P=0.013), which suggests an increase in sensitivity to cytotoxic agents. Analyses of Rb pathway mechanistic differences among these groups revealed frequent RB1 genomic alterations such as RB1 allelic loss and/or large spanning deletions (83%) in the p16 homo/RB1- subgroups, also indicating that RB1 deletions are frequent in high-grade serous carcinoma. CCNE1 gene gains/amplifications were frequent in the p16 homogeneous/RB1+ subgroup (68%) and cyclin D1 protein overexpression was predominantly characteristic of the p16 heterogeneous/RB1+ subgroup. These subcategories occur early in tumor progression and are seen with similar frequency in the cancer precursor lesion, serous tubal intra-epithelial carcinoma. Overall, this study uniquely identifies multiple non-synonymous mechanisms of retinoblastoma pathway deregulation that correlate with significantly different clinical outcomes. Furthermore, deregulations identified in precursor lesions suggest a key role of this pathway in serous tumor development. Recognition of these categories may identify patients with increased sensitivity to chemotherapy and new opportunities for novel therapeutics.

BRCA1 interacts with Nrf2 to regulate antioxidant signaling and cell survival.

Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.

Matrix metalloproteinase induction of Rac1b, a key effector of lung cancer progression.

Lung cancer is more deadly than colon, breast, and prostate cancers combined, and treatment improvements have failed to improve prognosis significantly. Here, we identify a critical mediator of lung cancer progression, Rac1b, a tumor-associated protein with cell-transforming properties that are linked to the matrix metalloproteinase (MMP)-induced epithelial-mesenchymal transition (EMT) in lung epithelial cells. We show that expression of mouse Rac1b in lung epithelial cells of transgenic mice stimulated EMT and spontaneous tumor development and that activation of EMT by MMP-induced expression of Rac1b gave rise to lung adenocarcinoma in the transgenic mice through bypassing oncogene-induced senescence. Rac1b is expressed abundantly in stages 1 and 2 of human lung adenocarcinomas and, hence, is an attractive molecular target for the development of new therapies that prevent progression to later-stage lung cancers.

CD36 repression activates a multicellular stromal program shared by high mammographic density and tumor tissues.

UNLABELLED: Although high mammographic density is considered one of the strongest risk factors for invasive breast cancer, the genes involved in modulating this clinical feature are unknown. Tissues of high mammographic density share key histologic features with stromal components within malignant lesions of tumor tissues, specifically low adipocyte and high extracellular matrix (ECM) content. We show that CD36, a transmembrane receptor that coordinately modulates multiple protumorigenic phenotypes, including adipocyte differentiation, angiogenesis, cell-ECM interactions, and immune signaling, is greatly repressed in multiple cell types of disease-free stroma associated with high mammographic density and tumor stroma. Using both in vitro and in vivo assays, we show that CD36 repression is necessary and sufficient to recapitulate the above-mentioned phenotypes observed in high mammographic density and tumor tissues. Consistent with a functional role for this coordinated program in tumorigenesis, we observe that clinical outcomes are strongly associated with CD36 expression. SIGNIFICANCE: CD36 simultaneously controls adipocyte content and matrix accumulation and is coordinately repressed in multiple cell types within tumor and high mammographic density stroma, suggesting that activation of this stromal program is an early event in tumorigenesis. Levels of CD36 and extent of mammographic density are both modifiable factors that provide potential for intervention.

p16(INK4a) expression and breast cancer risk in women with atypical hyperplasia.

p16, a nuclear protein encoded by the p16(INK4a) gene, is a regulator of cell-cycle regulation. Previous studies have shown that expression of p16 in tissue biopsies of patients with ductal carcinoma in situ (DCIS) is associated with increased risk of breast cancer, particularly when considered in combination with other markers such as Ki-67 and COX-2. Here, we evaluated how expression of p16 in breast tissue biopsies of women with atypical hyperplasia (AH), a putative precursor lesion to DCIS, is associated with subsequent development of cancer. p16 expression was assessed by immunohistochemistry in archival sections from 233 women with AH diagnosed at the Mayo Clinic. p16 expression in the atypical lesions was scored by percentage of positive cells and intensity of staining. We also studied coexpression of p16, with Ki-67 and COX-2, biomarkers of progression in AH. Risk factor and follow-up data were obtained via study questionnaire and medical records. Forty-seven patients (20%) developed breast cancer with a median follow-up of 14.5 years. Staining of p16 was increased in older patients relative to younger patients (P = 0.0025). Although risk of developing breast cancer was not associated with increased p16 expression, joint overexpression of Ki-67 and COX-2 was found to convey stronger risk of breast cancer in the first 10 years after diagnosis as compared with one negative marker (P < 0.01). However, the addition of p16 levels did not strengthen this association. p16 overexpression, either alone or in combination with COX-2 and Ki-67, does not significantly stratify breast cancer risk in women with AH.

Biomarker expression and risk of subsequent tumors after initial ductal carcinoma in situ diagnosis.

BACKGROUND: Studies have failed to identify characteristics of women who have been diagnosed with ductal carcinoma in situ (DCIS) and have a high or low risk of subsequent invasive cancer. METHODS: We conducted a nested case-control study in a population-based cohort of 1162 women who were diagnosed with DCIS and treated by lumpectomy alone from 1983 to 1994. We collected clinical characteristics and information on subsequent tumors, defined as invasive breast cancer or DCIS diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a regional or distant site greater than 6 months after initial treatment of DCIS (N = 324). We also conducted standardized pathology reviews and immunohistochemical staining for the estrogen receptor (ER), progesterone receptor, Ki67 antigen, p53, p16, epidermal growth factor receptor-2 (ERBB2, HER2/neu oncoprotein), and cyclooxygenase-2 (COX-2) on the initial paraffin-embedded DCIS tissue. Competing risk models were used to determine factors associated with risk of subsequent invasive cancer vs DCIS, and cumulative incidence survival functions were used to estimate 8-year risk. RESULTS: Factors associated with subsequent invasive cancer differed from those associated with subsequent DCIS. Eight-year risk of subsequent invasive cancer was statistically significantly (P = .018) higher for women with initial DCIS lesions that were detected by palpation or that were p16, COX-2, and Ki67 triple positive (p16(+)COX-2(+)Ki67(+)) (19.6%, 95% confidence interval [CI] = 18.0% to 21.3%) than for women with initial lesions that were detected by mammography and were p16, COX-2, and Ki67 triple negative (p16(-)COX-2(-)Ki67(-)) (4.1%, 95% CI = 3.4% to 5.0%). In a multivariable model, DCIS lesions that were p16(+)COX-2(+)Ki67(+) or those detected by palpation were statistically significantly associated with subsequent invasive cancer, but nuclear grade was not. Eight-year risk of subsequent DCIS was highest for women with DCIS lesions that had disease-free margins of 1 mm or greater combined with either ER(-)ERBB2(+)Ki67(+) or p16(+)COX-2(-)Ki67(+) status (23.6%, 95% CI = 18.1% to 34.0%). CONCLUSION: Biomarkers can identify which women who were initially diagnosed with DCIS are at high or low risk of subsequent invasive cancer, whereas histopathology information cannot.

Premalignant breast neoplasia: a paradigm of interlesional and intralesional molecular heterogeneity and its biological and clinical ramifications.

As is well established in invasive breast disease, it is becoming increasingly clear that molecular heterogeneity, both between and within lesions, is a prevalent, distinct phenotype of premalignant lesions of the breast. Key pathways of tumorigenesis modulate critical features of premalignant lesions such as proliferation, differentiation, stress response, and even the generation of diversity. Current studies show that evaluation of these lesions may provide clinically useful information on future tumor formation as well as biological insights into the origin and functional significance of this distinct phenotype.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy.

INTRODUCTION: We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells. METHODS: HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR). RESULTS: vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues. CONCLUSIONS: We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Abrogated response to cellular stress identifies DCIS associated with subsequent tumor events and defines basal-like breast tumors.

Approximately 15%-30% of women diagnosed with ductal carcinoma in situ (DCIS) develop a subsequent tumor event within 10 years after surgical lumpectomy. To date, little is known about the molecular pathways that confer this differential risk for developing subsequent disease. In this study, we demonstrate that expression of biomarkers indicative of an abrogated response to cellular stress predicts DCIS with worse outcome and is a defining characteristic of basal-like invasive tumors. Mechanistic studies identify the Rb pathway as a key regulator of this response. Conversely, biomarkers indicative of an intact response to cellular stress are strongly associated with a disease-free prognosis. Assessment of these biomarkers in DCIS begins to allow prediction of tumor formation years before it actually occurs.

p16INK4a modulates p53 in primary human mammary epithelial cells.

p16(INK4a) (p16) and p53 are tumor suppressor genes that are inactivated during carcinogenesis in many tumors. Here we show that p16 gene activity inversely modulates p53 status and function in primary human mammary epithelial cells. Reduced levels of p16 protein stabilize p53 protein through inhibition of proteolytic degradation, and this increase in p53 protein levels enhances the cellular response to radiation, represses proliferation, and transcriptionally activates downstream targets. Stabilization of p53 is mediated through the retinoblastoma/E2F/p14(ARF)/murine double minute-2 pathway. However, we have observed that p16 does not modulate p53 in fibroblasts, indicating a possible cell type-specific regulation of this pathway.

p38 regulates cyclooxygenase-2 in human mammary epithelial cells and is activated in premalignant tissue.

The immediate-early gene, cyclooxygenase-2 (COX-2), is induced in a variety of inflammatory and neoplastic processes and is believed to play an important role in tumorigenesis. In this study, we identify an important upstream regulatory pathway of COX-2 expression in variant human mammary epithelial cells (vHMEC), which has been shown to exhibit phenotypes important for malignancy. We find that the stress-activated kinase, p38, is phosphorylated and activated in vHMEC compared with HMEC and is responsible for the expression of COX-2 in vHMEC as cells grow in culture. Furthermore in this capacity, p38 acts to stabilize the COX-2 transcript rather than activate COX-2 transcription. Inhibition of p38 kinase, using a chemical inhibitor, down-regulates COX-2 and decreases cell survival. Examination of archived tissue from women with ductal carcinoma in situ reveals epithelial cells that not only overexpress COX-2 but also have an abundance of activated phospho-p38 in the nucleus and cytoplasm, mirroring the expression observed in vitro. These epithelial cells are found within premalignant lesions as well as in fields of morphologically normal tissue that surround the lesions. In contrast, low phospho-p38 staining was observed in the majority of normal tissue obtained from reduction mammoplasty. These data help define the regulation of COX-2 expression in early carcinogenesis and provide alternative candidates for targeted prevention of COX-2-induced phenotypes and breast cancer.

Genetic and epigenetic changes in mammary epithelial cells may mimic early events in carcinogenesis.

Studies of human mammary epithelial cells from healthy individuals are providing novel insights into how early epigenetic and genetic events affect genomic integrity and fuel carcinogenesis. Key epigenetic changes, such as the hypermethylation of the p16 (INK4a) promoter sequences, create a previously unappreciated preclonal phase of tumorigenesis in which a subpopulation of mammary epithelial cells are positioned for progression to malignancy (Romanov et al. , 2001, Nature , 409:633-637; Tlsty et al. , 2001, J. Mammary Gland Biol. Neoplasia , 6:235-243). These key changes precede the clonal outgrowth of premalignant lesions and occur frequently in healthy, disease-free women. Understanding more about these early events should provide novel molecular candidates for prevention and therapy of breast cancer that target the process instead of the consequences of genomic instability. This review will highlight some of the key alterations that have been studied in human mammary epithelial cells in culture and relate them to events observed in vivo and discussed in accompanying reviews in this volume.

Histologically normal human mammary epithelia with silenced p16(INK4a) overexpress COX-2, promoting a premalignant program.

Breast tissue from healthy women contains variant mammary epithelial cells (vHMEC) exhibiting p16INK4a promoter hypermethylation both in vivo and in vitro. When continuously cultured, vHMEC acquire telomeric dysfunction and produce the types of chromosomal abnormalities seen in premalignant lesions of cancer. We find that late passage vHMEC express elevated prostaglandin cyclo-oxygenase 2 (COX-2), which contributes to increased prostaglandin synthesis, angiogenic activity, and invasive ability. These data demonstrate the existence of human mammary epithelial cells with the potential to acquire multiple genomic alterations and phenotypes associated with malignant cells. Moreover, COX-2 overexpression coincides with focal areas of p16INK4a hypermethylation in vivo, creating ideal candidates as precursors to breast cancer. These putative precursors can be selectively eliminated upon exposure to COX-2 inhibitors in vitro.

Protein kinase Calpha negatively regulates cell spreading and motility in MDA-MB-231 human breast cancer cells downstream of epidermal growth factor receptor.

Previous work has shown that phorbol esters modulate chemotaxis. Here, we demonstrate that PKC activation via phorbol 12-myristate 13-acetate (PMA) treatment of MDA-MB-231 cells inhibits EGF-induced cell spreading, the initial event of motility and chemotaxis. Of five PKC isoforms (alpha,iota,lambda,delta,and epsilon) identified in this cell line, PMA treatment only induced PKCalpha translocation from the cytosol to the membrane, an event that correlated with the development of the rounded morphology. Cell recovery was linked to PKCalpha downregulation in part via the proteasome pathway since treatment with MG101 in the presence of PMA did not lead to PKCalpha degradation and cell recovery. Co-immunoprecipitation and immunolocalization demonstrated that EGF co-localized with PKCalpha and EGFR, however, PMA did not abrogate EGFR transactivation. This work suggests that PKCalpha is the primary target of PMA acting as a transient negative regulator of cell spreading and motility in MDA-MB-231 breast cancer cells.

Cyclooxygenase-2 expression is related to nuclear grade in ductal carcinoma in situ and is increased in its normal adjacent epithelium.

Cyclooxygenase-2 (COX-2) is emerging as an important cancer biomarker and is now an experimental target for solid tumor treatment.However, no study has exclusively focused on COX-2 expression in early lesions such as ductal carcinoma in situ (DCIS). We examined COX-2 expression by immunohistochemistry in 46 cases of women undergoing surgical resection for DCIS. We found that COX-2 expression was detected in 85% of all DCIS specimens, with increased COX-2 staining correlating with higher nuclear grade. Strikingly, COX-2 staining intensity in the normal adjacent epithelium was stronger than in the DCIS lesion itself. Our observations demonstrate that COX-2 is up-regulated in the normal adjacent epithelium and supports the hypothesis that the surrounding epithelial tissue is part of the disease process in DCIS.

Post-mortem changes in calmodulin binding proteins in muscle and lung.

Estimation of post-mortem interval (PMI) remains an elusive issue in forensic investigations. In this study, we examined the possible use of calmodulin (CaM) binding proteins (CaMBPs) as indicators of PMI. Whole CaMBP populations from homogenized rat lung and rat skeletal muscle removed at 0, 24, 48 and 96 h post-mortem at 21 degrees C were detected by the calmodulin binding overlay technique (CaMBOT) using 35S-VU1-CaM and visualized by autoradiography. CaMBOT showed that, in both tissues, the CaMBP population remained relatively stable for up to 96 h post-mortem with the exception of a single approximately 200 kDa CaMBP that increased in 24 h post-mortem samples then showed decreasing amounts at subsequent times. Immunoblot analysis of the specific CaMBPs, Ca(2+)/CaM-dependent kinase II (CaMKII), calcineurin A (CNA), myristoylated alanine-rich C-kinase substrate (MARCKS) and inducible nitric oxide synthase (iNOS) were done on lung tissue samples. CaMKII levels did not change appreciably over the 96 h PMI examined. In contrast to iNOS levels, which varied from sample to sample, CNA and MARCKS showed predictable patterns of change: the level of MARCKS decreased steadily in the 0-96 h post-mortem lung samples while CNA underwent a shift in mobility on SDS-PAGE by 24 h post-mortem before slowly decreasing in amount. The stability of CaMKII levels over 96 h was also seen in skeletal muscle tissue while CNA showed variable levels at 0, 48 and 96 h with the presence of the rapidly migrating band at 24 h. These patterns of change in CaMBPs provide some insight into the post-mortem changes in calmodulin-mediated signaling components in lung and skeletal muscle and support the further study of CNA and CaMKII as potential markers for estimating short- and long-term PMIs.