Maintenance of hormone responsiveness in luminal breast cancers by suppression of Notch.
ABSTRACT: Luminal breast cancers express estrogen (ER) and/or progesterone (PR) receptors and respond to hormone therapies. Basal-like "triple negative" cancers lack steroid receptors but are cytokeratin (CK) 5-positive and require chemotherapy. Here we show that more than half of primary ER(+)PR(+) breast cancers contain an ER(-)PR(-)CK5(+) "luminobasal" subpopulation exceeding 1% of cells. Starting from ER(+)PR(+) luminal cell lines, we generated lines with varying luminal to luminobasal cell ratios and studied their molecular and biological properties. In luminal disease, luminobasal cells expand in response to antiestrogen or estrogen withdrawal therapies. The phenotype and gene signature of the hormone-resistant cells matches that of clinical triple negative basal-like and claudin-low disease. Luminobasal cell expansion in response to hormone therapies is regulated by Notch1 signaling and can be blocked by ?-secretase inhibitors. Our data establish a previously unrecognized plasticity of ER(+)PR(+) luminal breast cancers that, without genetic manipulation, mobilizes outgrowth of hormone-resistant basal-like disease in response to treatment. This undesirable outcome can be prevented by combining endocrine therapies with Notch inhibition.
Project description:Luminal breast cancers express estrogen (ER) and/or progesterone (PR) receptors and respond to hormone therapies. Basal-like “triple negative” (TN) cancers lack steroid receptors but are cytokeratin (CK) 5-positive and require chemotherapy. Here we show that over half of primary ER+PR+ breast cancers contain an ER–PR–CK5+ “luminobasal” subpopulation exceeding 1% of cells. Starting from ER+PR+ luminal cell lines, we generated novel lines with varying luminal to luminobasal-cell ratios and studied their molecular and biological properties. In luminal disease, luminobasal cells expand in response to antiestrogen or estrogen withdrawal therapies. The phenotype and gene signature of the hormone resistant cells matches that of clinical TN basal-like and claudin-low disease. Luminobasal-cell expansion in response to hormone therapies is regulated by Notch1 signaling and can be blocked by gamma-secretase inhibitors (GSI). Our data establish a previously unrecognized plasticity of ER+PR+ luminal breast cancers that, without genetic manipulation, mobilizes outgrowth of hormone-resistant basal-like disease in response to treatment. This undesirable outcome can be prevented by combining endocrine therapies with Notch inhibition. 24 array samples
Project description:<h4>Introduction</h4>Luminal, estrogen receptor-positive (ER(+)) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models.<h4>Methods</h4>Immunocompromised, ovariectomized (ovx'd) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER(+)PR(+) cytokeratin 5-negative (CK5(-)) E3 and MCF-7 cells, basal ER(-)PR(-)CK5(+) estrogen withdrawn-line 8 (EWD8) cells, and basal ER(-)PR(-)CK5(-) MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx'd mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers.<h4>Results</h4>ER(-)PR(-) cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx'd mice injected with ER(+)PR(+) cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER(+)PR(+)CK5(-) cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER(-)PR(-)CK5(+) cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx'd control mice injected with ER(+)PR(+) cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER(+) but PR(-). Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration.<h4>Conclusions</h4>We conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER(-)PR(-) subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx'd mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes.
Project description:Epidemiologic studies suggest that some hormone-related risk factors in breast cancer differentially influence risk for disease subtypes classified by the status of the estrogen and progesterone receptors (ER/PR). However, it remains unclear whether human epidermal growth factor receptor 2 (HER2) or p53 expression status further differentiates these exposure-risk group associations. We evaluated the associations of oral contraceptive (OC) use and reproductive factors with incident invasive breast cancer subtypes among 1,197 population-based cases and 2,015 controls from the Los Angeles County or Detroit components of the Women's Contraceptive and Reproductive Experiences Study. Case-control comparisons by ER/PR/HER2/p53 status were conducted by multivariable polychotomous unconditional logistic regression methods. We found that OC use was not associated with any breast cancer subtype as defined by ER/PR/HER2/p53 status, except for a 2.9-fold increased risk of so-called triple-negative tumors (ER(-)/PR(-)/HER2(-)) among women of 45 to 64 years of age who started OC use before age 18. Parity was associated with a decreased risk of luminal A (ER(+) or PR(+), HER2(-)), luminal B (ER(+) or PR(+)/HER2(+)), and ER(-)/PR(-)/HER2(+) tumors. Age at first full-term pregnancy was positively associated with luminal A tumors among older women. Neither of these reproductive factors was associated with triple-negative tumors. Long duration of breast-feeding lowered the risk of triple-negative and luminal A tumors. p53 status did not define further differential risk patterns. Our findings offer evidence of differences in the hormone-related risk factors between triple-negative cancers and other ER/PR/HER2-defined subtypes of breast cancer.
Project description:There are two major subtypes of human breast cancers: the luminal, estrogen, and progesterone receptor-positive, cytokeratin 18-positive (ER(+)PR(+)CK18(+)) subtype, and the basal ER(-)PR(-)CK18(-)CK5(+) subtype. Tumor-initiating cells (CD44(+)) have been described for human breast cancers; whether these are common to the two subtypes is unknown. We have identified a rare population of cells that are both CD44(+) and ER(-)PR(-)CK5(+) in luminal-like ER(+)PR(+) T47D human breast tumor xenografts. The tumor-isolated CD44(+) cell fraction was highly enriched for clonogenic (in vitro culture) and tumorigenic (in vivo reimplantation) cells compared with the CD44(-) cell fraction. Rare ER(-)PR(-)CK5(+) cells were present within CD44(+)-derived colonies. Tumor-isolated cells placed in minimal media also contained rare ER(-)PR(-)CK5(+) cells at early time points (<10 cells); however, this population did not expand with increasing colony size. The number of ER(+)PR(+)CK5(-) cells, conversely, increased linearly with colony growth. Similary, tumors originating in vivo from CD44(+) cells contained a rare static ER(-)PR(-)CK5(+) population, an intermediate ER(-)PR(-)CK5(-) population, and an expanding ER(+)PR(+)CK5(-) population. Putative ER(+)PR(+)CK5(+) transitional cells could be seen only in colonies or tumors treated with a progestin. We propose that luminal ER(+)PR(+) breast tumors contain a minor ER(-)PR(-)CK5(+) population that has the capacity to generate the majority of ER(+)PR(+)CK18(+)CK5(-) cells. Luminal breast cancers are treated with endocrine therapies that target ER. The rare ER(-)PR(-)CK5(+) progenitor cells would escape such treatments and survive to repopulate the tumor.
Project description:Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as "triple negative breast cancer" (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis.
Project description:Human primary breast cancers and breast cancer cell lines are classified by microarray-defined molecular subtypes, which reflect differentiation characteristics. Estrogen receptor (ER) expression is indicative of the luminal molecular subtype. We have previously established IPH-926, the first well-characterized cell line from infiltrating lobular breast cancer. IPH-926 displays an ER/PR/ErbB2 triple-negative immunophenotype, which is due to a loss of ER expression in its in vivo clonal ancestry. Loss of ER might indicate a fundamental change of cellular differentiation and it is unclear whether a luminal subtype is preserved beyond ER conversion. Using Affymetrix microarray analysis, seven different classifier gene lists (PAM305, DISC256, TN1288, PAM50, UNC1300, LAB704, INT500) and a background population of 50 common mammary carcinoma cell lines, we have now determined the molecular subtype of IPH-926. Strikingly, the IPH-926 expression profile is highly consistent with a luminal subtype. It is nearest to luminal/ER-positive breast cancer cell lines and far apart from basal breast cancer cell lines. Quantitative real-time RT-PCR confirmed enhanced expression of luminal marker genes (AGR2, CLU, CA12, EMP2, CLDN3) and low or absent expression of basal marker genes (KRT5, CD44, CAV1, VIM). Moreover, IPH-926 lacked androgen receptor (AR) expression, a transcription factor previously associated with luminal-like gene expression in a subset of triple-negative or molecular apocrine breast cancers. In conclusion, IPH-926 is triple-negative but belongs to the luminal subtype. Luminal differentiation characteristics can be preserved beyond ER conversion and might not require a compensatory expression of AR.
Project description:Many Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+ PR+ ones. One such subpopulation we call "Luminobasal" is ER-, PR- and cytokeratin 5 (CK5)-positive. It is not targeted for treatment.To address the relationships between ER+PR+CK5- and ER-PR-CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We used high-throughput screening to identify pLB-specific drugs and examined their efficacy alone and in combination with hormone therapy in mixed-cell tumor models.We show that pLUM and MCF7 cells suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and that pLUM cells suppress growth of pLB cells in mixed-cell xenografts in vivo. High-throughput screening of 89 FDA-approved oncology drugs shows that pLB cells are sensitive to monotherapy with the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib. By exploiting mixed-cell 3D colonies and mixed-cell solid mouse tumors models we demonstrate that combination therapy with gefitinib plus the anti-estrogen fulvestrant constitutes a robust treatment strategy.We propose that response to combination endocrine/EGFR inhibitor therapies in heterogeneous Luminal cancers may improve long-term survival in patients whose primary tumors have been preselected for appropriate biomarkers, including ER, PR, CK5 and EGFR.
Project description:The relation between breast cancer molecular subtype and survival has been studied in several jurisdictions, but limited information is available for Ontario. The aim of this study was to determine breast cancer survival by molecular subtype and to assess the effect on survival of selected demographic and tumour-based characteristics.We extracted 29?833 breast cancer cases (in 26?538 girls and women aged ? 15 yr) diagnosed between 2010 and 2012 from the Ontario Cancer Registry. Cancers were categorized into 4 molecular subtypes: 1) luminal A (estrogen-receptor-positive and/or progesterone-receptor-positive [ER+ and/or PR+] and negative for human epidermal growth factor receptor 2 [HER2-]), 2) luminal B (ER+ and/or PR+/HER2+), 3) HER2-enriched (ER- and PR-/HER2+) and 4) triple-negative (ER- and PR-/HER2-). We estimated associations with predictor variables (age, stage at diagnosis, histologic type, comorbidity and place of residence [urban or rural]) using a multivariate Cox proportional hazards model. Likelihood ratio testing was used to evaluate differences in risk of death.Luminal A was the most commonly diagnosed subtype (59.0%) and had the greatest survival, whereas triple-negative had the poorest survival. For all subtypes, a dose-response effect was observed between the hazard of death and age and stage at diagnosis, with the greatest effect found for the HER2-enriched subtype (age: hazard ratio [HR] 7.87 [95% confidence interval (CI) 3.68-11.81]; stage at diagnosis: HR 37.71 [95% CI 34.64-41.27]). Moderate comorbidity (Charlson Comorbidity Index score 1 or 2) was associated with increased risk of death for triple-negative cancers (HR 2.42 [95% CI 1.36-4.31]), and severe comorbidity (Charlson Comorbidity Index score ? 3) increased the risk for all molecular subtypes.The results indicate the importance of including molecular subtype, along with age, stage at diagnosis and comorbidity, in assessing breast cancer survival. They highlight the need to address outcomes related to hormone-receptor-negative cancers, for which survival lags behind that for hormone-receptor-positive cancers.
Project description:BACKGROUND:Epidemiologic data suggest that parity increases risk of hormone receptor-negative breast cancer and that breastfeeding attenuates this association. Prospective data, particularly on the joint effects of higher parity and breastfeeding, are limited. METHODS:We investigated parity, breastfeeding, and breast cancer risk by hormone-receptor (estrogen (ER) and progesterone receptor (PR)) and molecular subtypes (luminal A, luminal B, HER2-enriched, and basal-like) in the Nurses' Health Study (NHS; 1976-2012) and NHSII (1989-2013). A total of 12,452 (ER+ n = 8235; ER- n = 1978) breast cancers were diagnosed among 199,514 women. We used Cox proportional hazards models, adjusted for breast cancer risk factors, to calculate hazard ratios (HR) and 95% confidence intervals (CI). RESULTS:Parous women had lower risk of ER+ breast cancer (vs. nulliparous, HR = 0.82 [0.77-0.88]); no association was observed for ER- disease (0.98 [0.84-1.13]; Phet = 0.03). Among parous women, breastfeeding was associated with lower risk of ER- (vs. never 0.82 [0.74-0.91]), but not ER+, disease (0.99 [0.94-1.05]; Phet < 0.001). Compared to nulliparous women, higher parity was inversely associated with luminal B breast cancer regardless of breastfeeding (≥ 3 children: ever breastfed, 0.78 [0.62-0.98]; never breastfed, 0.76 [0.58-1.00]) and luminal A disease only among women who had breastfed (≥ 3 children, 0.84 [0.71-0.99]). Basal-like breast cancer risk was suggestively higher among women with higher parity who never breastfed; associations were null among those who ever breastfed. CONCLUSIONS:This study provides evidence that breastfeeding is inversely associated with hormone receptor-negative breast cancers, representing an accessible and cost-effective risk-reduction strategy for aggressive disease subtypes.
Project description:BACKGROUND/AIM:Mast cells (MCs) represent the most controversial non-malignant element of the tumor microenvironment. Our aim was to study how MCs density and distribution (intratumoral-MCit versus peritumoral-MCpt) relate to tumor grade and molecular subtypes. MATERIALS AND METHODS:MCs tryptase immunohistochemistry was performed on 80 cases of breast carcinomas. RESULTS:For Luminal A tumors, a partial correlation was detected between MCit and progesterone receptor (PR) (p=0.005). Luminal B tumors showed a significant correlation between MCpt and age (p=0.009), estrogen receptor (ER) (p=0.017) and PR (p=0.035). MCit and MCpt were strongly interrelated in this subtype (p=0.002) and in triple-negative breast cancers (p=0.002). In HER2 subtype, MCpt tumors were significantly correlated with HER2 (p=0.044). In G2 tumors, MCpt correlated with ER (p=0.015) and PR (p=0.038) while in G3 tumors ER correlated with both MCit (p=0.009) and MCpt (p=0.000487) tumors. CONCLUSION:MCs dynamics are strongly influenced by hormone receptors and HER2 status. MCit increased in aggressive tumor types and is a worse prognostic factor.