Haoheng Yan

A Genome-wide siRNA Screen Identifies Proteasome Addiction as a Vulnerability of Basal-like Triple-Negative Breast Cancer Cells

Basal-like triple-negative breast cancers (TNBCs) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes: basal-like BPLER and myoepithelial HMLER. Expression of the screens 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal, and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence.

Stability, toxicity and differential cellular uptake of protein passivated-Fe3O4 nanoparticles

We have explored the mechanism and differential uptake of BSA coated Fe3O4nanoparticles (NPs) by different cancerous and isogenic cell types.

Repression of mammary stem/progenitor cells by p53 is mediated by notch and separable from apoptotic activity

Breast cancer is the most common tumor among women with inherited mutations in the p53 gene (Li‐Fraumeni syndrome). The tumors represent the basal‐like subtype, which has been suggested to originate from mammary stem/progenitor cells. In mouse mammary epithelium, mammosphere‐forming potential was increased with decreased dosage of the gene encoding the p53 tumor suppressor protein (Trp53). Limiting dilution transplantation also showed a 3.3‐fold increase in the frequency of long‐term regenerative mammary stem cells in Trp53−/− mice. The repression of mammospheres by p53 was apparent despite the absence of apoptotic responses to radiation indicating a dissociation of these two activities of p53. The effects of p53 on progenitor cells were also observed in TM40A cells using both mammosphere‐forming assays and the DsRed‐let7c‐sensor. The frequency of long‐term label‐retaining epithelial cells was decreased in Trp53−/− mammary glands indicating that asymmetric segregation of DNA is diminished and contributes to the expansion of the mammary stem cells. Treatment with an inhibitor of γ‐secretase (N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester) reduced the number of Trp53−/− mammospheres to the level found in Trp53+/+ cells. These results demonstrate that basal levels of p53 restrict mammary stem/progenitor cells through Notch and that the Notch pathway is a therapeutic target to prevent expansion of this vulnerable pool of cells. STEM CELLS 2011;29:119–127

Transcriptional Responses to Estrogen and Progesterone in Mammary Gland Identify Networks Regulating p53 Activity

Estrogen and progestins are essential for mammary growth and differentiation but also enhance the activity of the p53 tumor suppressor protein in the mammary epithelium. However, the pathways by which these hormones regulate p53 activity are unknown. Microarrays were used to profile the transcriptional changes within the mammary gland after administration of either vehicle, 17beta-estradiol (E), or progesterone (P) individually and combined (EP). Treatment with EP yielded 1182 unique genes that were differentially expressed compared to the vehicle-treated group. Although 30% of genes were responsive to either E or P individually, combined treatment with both EP had a synergistic effect accounting for 60% of the differentially regulated genes. Analysis of protein-protein interactions identified p53, RelA, Snw1, and Igfals as common targets of genes regulated by EP. RelA and p53 form hubs within a network connected by genes that are regulated by EP and that may coordinate the competing functions of RelA and p53 in proliferation and survival of cells. Induction of early growth response 1 (Egr1) and Stratifin (Sfn) (also known as 14-3-3sigma) by EP was confirmed by reverse transcription-quantitative PCR and shown to be p53 independent. In luciferase reporter assays, Egr1 was shown to enhance transcriptional activation by p53 and inhibit nuclear factor kappaB activity. These results identify a gene expression network that provides redundant activation of RelA to support proliferation as well as sensitize p53 to ensure proper surveillance and integration of their competing functions through factors such as Egr1, which both enhance p53 and inhibit RelA.

Estrogen and progesterone induce persistent increases in p53-dependent apoptosis and suppress mammary tumors in BALB/c-Trp53+/- mice

IntroductionTreatment with estrogen and progesterone (E+P) mimics the protective effect of parity on mammary tumors in rodents and depends upon the activity of p53. The following experiments tested whether exogenous E+P primes p53 to be more responsive to DNA damage and whether these pathways confer resistance to mammary tumors in a mouse model of Li-Fraumeni syndrome.MethodsMice that differ in p53 status (Trp53+/+, Trp53+/-, Trp53-/-) were treated with E+P for 14 days and then were tested for p53-dependent responses to ionizing radiation. Responses were also examined in parous and age-matched virgins. The effects of hormonal exposures on tumor incidence were examined in BALB/c-Trp53+/- mammary tissues.ResultsNuclear accumulation of p53 and apoptotic responses were increased similarly in the mammary epithelium from E+P-treated and parous mice compared with placebo and age-matched virgins. This effect was sustained for at least 7 weeks after E+P treatment and did not depend on the continued presence of ovarian hormones. Hormone stimulation also enhanced apoptotic responses to ionizing radiation in BALB/c-Trp53+/- mice but these responses were intermediate compared with Trp53+/+ and Trp-/- tissues, indicating haploinsufficiency. The appearance of spontaneous mammary tumors was delayed by parity in BALB/c-Trp53+/- mice. The majority of tumors lacked estrogen receptor (ER), but ER+ tumors were observed in both nulliparous and parous mice. However, apoptotic responses to ionizing radiation and tumor incidence did not differ among outgrowths of epithelial transplants from E+P-treated donors and nulliparous donors.ConclusionTherefore, E+P and parity confer a sustained increase in p53-mediated apoptosis within the mammary epithelium and suppress mammary tumorigenesis, but this effect was not retained in epithelial outgrowths.

Cleaning of nanoparticle inhibitors via proteolysis of adsorbed proteins

Cytochrome c adsorbed to anionic nanoparticles is selectively proteolyzed by trypsin, providing a mechanism for the catalytic degradation of proteins.

Regulation of cancer stem cells by p53.

The hypothesis that cancer stem cells are responsible for the chemoresistant and metastatic phenotypes of many breast cancers has gained support using cell-sorting strategies to enrich the tumor-initiating population of cells. The mechanisms regulating the cancer stem cell pool, however, are less clear. Two recent publications suggest that loss of p53 permits expansion of presumptive cancer stem cells in mouse mammary tumors and in human breast cell lines. These results add restriction of cancer stem cells as a new tumor suppressor activity attributed to p53.

Pathways Contributing to Development of Spontaneous Mammary Tumors in BALB/c-Trp53+/− Mice

Mutation and loss of function in p53 are common features among human breast cancers. Here we use BALB/c-Trp53+/- mice as a model to examine the sequence of events leading to mammary tumors. Mammary gland proliferation rates were similar in both BALB/c-Trp53+/- mice and wild-type controls. In addition, sporadic mammary hyperplasias were rare in BALB/c-Trp53+/- mice and not detectably different from those of wild-type controls. Among the 28 mammary tumors collected from BALB/c-Trp53+/- mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53+/- ductal hyperplasias also indicated an association between loss of the wild-type allele of Trp53 and progression to invasive carcinomas. Therefore, loss of p53 function seems to be a rate-limiting step in progression. Moreover, expression of biomarkers such as estrogen receptor alpha, progesterone receptor, Her2/Neu, and activated Notch1 varied among mammary tumors, suggesting that multiple oncogenic lesions collaborate with loss of p53 function. Expression of biomarkers was retained when tumor fragments were transplanted to syngeneic hosts. Tumors expressing solely luminal or basal keratins were also observed (27 and 11%, respectively), but the largest class of tumors expressed both luminal and basal keratins (62%). Overall, this panel of transplantable tumors provides a resource for detailed evaluation of the cell lineages undergoing transformation and preclinical testing of therapeutic agents targeting a variety of oncogenic pathways including cancer stem cells.

Abstract SY43-03: Screening for triple negative breast cancer vulnerabilities

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA There is no targeted therapy for triple negative breast cancers (TNBC), which have the worst prognosis of human breast cancers. TNBCs are prone to relapse and metastasize after cytotoxic drug treatment. This group of cancers, defined by low or absent expression of estrogen, progesterone and Her2 receptors, are heterogeneous in genetic, epigenetic and phenotypic features, making it difficult to identify specific drug targets suitable for this group of cancers as a whole. About half or more of TNBCs have an epithelial phenotype (classified based on gene expression profiling as basal-like or basal-A) and a large minority of the remaining tumors are mesenchymal (classified as basal-B). To identify selective genetic dependencies of the basal-like subtype of TNBC that are not required for survival of normal breast epithelial cells, which might be good targets for targeted drug therapy, we first performed a genome-wide siRNA lethality screen that compared two human breast epithelial TNBC cell lines transformed with the same genes in different media - basal-like BPLER and myoepithelial HMLER. BPLER are highly malignant and enriched for tumor-initiating cells, forming tumors in nude mice after injection of just 50 cells, while the more differentiated HMLER cell line only forms tumors when more than 50,000 cells are injected. The screen identified 154 genes on which BPLER, but not HMLER, depended. Expression of the 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Of note, no oncogenic kinases, which are often chosen as cancer drug targets, were identified in the screen. Proteasome genes were overrepresented hits. Basal-like TNBC lines as a group were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal breast cancer and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiation and metastasis. Tumor-initiating cells (T-ICs) within basal-like cancers did not survive proteasome inhibition. Developing ways to target T-ICs is a priority of cancer drug development. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence, which was also a common dependency gene of basal-like TNBCs. Because of the heterogeneity of TNBCs, we next assessed how many of the BPLER dependencies were shared with other breast cancers. A targeted screen was performed comparing the viability of 10 breast cancer cell lines (3 luminal A, 5 basal-A and 2 basal-B TNBCs) and telomerase-immortalized normal breast epithelial cells after knockdown of the 154 BPLER dependency genes. This screen confirmed the selective dependence of basal-A TNBCs on the proteasome and MCL-1. This screen identified 17 additional hits that were shared dependencies in at least 4 of 5 basal-like TNBC cell lines. A novel hit, required for survival of 4 of 5 basal-A TNBC cell lines, but not normal breast epithelial cells, was the spindle checkpoint regulator NDC80. This work was supported by Department of Defense grants to FP and JL and by a grant from the Harvard Stem Cell Institute. The authors have no financial conflicts of interest to disclose. Citation Format: Fabio Petrocca, Gabriel Altschuler, Shen Mynn Tan, Marc L. Mendillo, Haoheng Yan, D. Joseph Jerry, Andrew L. Kung, Winston Hide, Tan A. Ince, Judy Lieberman. Screening for triple negative breast cancer vulnerabilities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY43-03. doi:10.1158/1538-7445.AM2014-SY43-03

Abstract 3380: Transient Tgfβ exposure causes persistent transdifferentiation in mouse mammary epithelial cells in vitro and in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Transforming growth factor beta (Tgfβ) is transiently increased during involution in the mammary gland following a pregnancy and may promote the risk of pregnancy associated breast cancer (PABC). While tgfβ inhibits growth of normal mammary epithelium and has a functional role in restoring the pre-pregnancy architecture of the mammary gland, this cytokine also has tumor promoter function because it promotes epithelial to mesenchymal transition (EMT) and initiates metastatic behavior in tumor cells. We hypothesize that transient Tgfβ exposure, to mimic involution, selects for cells that are susceptible to EMT and increases risk for PABC. We have found that short term 14 day treatment of CDβGeo cells, a mouse mammary epithelial cell line, with Tgfβ (5ng/ml) promotes EMT. These cells are persistently transdifferentiated (pTD) even after withdrawal of Tgfβ. In contrast to the parental CDβGeo cells, the pTD cells have decreased expression of E-cadherin (3-fold, p 3-fold, p 5-fold;p<0.01). In addition, expression of Tgfb2 mRNA was also increased (2.8-fold;p<0.05) in the pTD cells. These results demonstrate that transient exposure to Tgfβ causes persistent transdifferentation with increases in Snail and Tgfb2 expression in CDβGeo cells and suggest maintenance of transdifferentiation through an autocrine positive feedback loop. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3380. doi:10.1158/1538-7445.AM2011-3380