Xiangshan Zhao

Histological, molecular and functional subtypes of breast cancers

Increased understanding of the molecular heterogeneity that is intrinsic to the various subtypes of breast cancer will likely shape the future of breast cancer diagnosis, prognosis, and treatment. Advances in the field over the last several decades have been remarkable and have clearly translated into better patient care as evidenced by the earlier detection, better prognosis, and new targeted therapies. There have been two recent advances in the breast cancer research field that have lead to paradigm shifts: first, the identification of intrinsic breast tumor subtypes, which has changed the way we think about breast cancer and second, the recent characterization of cancer stem cells (CSCs), which are suspected to be responsible for tumor initiation, recurrence and resistance to therapy, have opened new exciting avenues to think about breast cancer therapeutic strategies. While these advances constitute major paradigm shifts within the research realm, the clinical arena has yet to adopt and apply our understanding of the molecular basis of the disease to early diagnosis, prognosis and therapy of breast cancers. Here, we will review the current clinical approach to classification of breast cancers, newer molecular-based classification schemes, and potential future of biomarkers representing a functional classification of breast cancer.

Anticancer activity of Celastrol in combination with ErbB2-targeted therapeutics for treatment of ErbB2-overexpressing breast cancers.

The receptor tyrosine kinase ErbB2 is overexpressed in up to a third of breast cancers, allowing targeted therapy with ErbB2-directed humanized antibodies such as Trastuzumab. Concurrent targeting of ErbB2 stability with HSP90 inhibitors is synergistic with Trastuzumab, suggesting that pharmacological agents that can inhibit HSP90 as well as signaling pathways activated by ErbB2 could be useful against ErbB2-overexpressing breast cancers. The triterpene natural product Celastrol inhibits HSP90 and several pathways relevant to ErbB2-dependent oncogenesis including the NFκB pathway and the proteasome, and has shown promising activity in other cancer models. Here, we demonstrate that Celastrol exhibits in vitro antitumor activity against a panel of human breast cancer cell lines with selectivity towards those overexpressing ErbB2. Celastrol strongly synergized with ErbB2-targeted therapeutics Trastuzumab and Lapatinib, producing higher cytotoxicity with substantially lower doses of Celastrol. Celastrol significantly retarded the rate of growth of ErbB2-overexpressing human breast cancer cells in a mouse xenograft model with only minor systemic toxicity. Mechanistically, Celastrol not only induced the expected ubiquitinylation and degradation of ErbB2 and other HSP90 client proteins, but it also increased the levels of reactive oxygen species (ROS). Our studies show that the Michael Acceptor functionality in Celastrol is important for its ability to destabilize ErbB2 and exert its bioactivity against ErbB2-overexpressing breast cancer cells. These studies suggest the potential use of Michael acceptor-containing molecules as novel therapeutic modalities against ErbB2-driven breast cancer by targeting multiple biological attributes of the driver oncogene.

Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types—a K5+/K19− type and a K5+/K19+ type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

Shared signaling pathways in normal and breast cancer stem cells.

Recent advances in our understanding of breast cancer biology have led to the identification of a subpopulation of cells within tumors that appear to be responsible for initiating and propagating the cancer. These tumor initiating cells are not only unique in their ability to generate tumors, but also share many similarities with elements of normal adult tissue stem cells, and have therefore been termed cancer stem cells (CSCs). These CSCs often inappropriately use many of the same signaling pathways utilized by their normal stem cell counterparts which may present a challenge to the development of CSC specific therapies. Here, we discuss three major stem cell signaling pathways (Notch, Wnt, and Hedgehog); with a focus on their function in normal mammary gland development and their misuse in breast cancer stem cell fate determination.

Cyclooxygenase-2 Expression during Immortalization and Breast Cancer Progression

Identification of molecular aberrations in premalignant human mammary epithelial cells (hMEC), the precursors for breast cancers, is a central goal in breast cancer biology. Recent studies implicated expression of cyclooxygenase 2 (COX-2) as a marker to identify precursor cells for breast cancer. In this study, we analyzed COX-2 expression in preselection and postselection hMEC cells and observed similar COX-2 levels in both cells. Interestingly, immortalization of postselection cells using various methods leads to a dramatic decrease in COX-2 expression. Similar to immortal cells, the majority of breast cancer cell lines expressed low levels of COX-2 protein. Finally, analyses of COX-2 expression in a series of specimens from reduction mammoplasty, adenosis, ductal carcinoma in situ, and infiltrating ductal carcinoma showed down-regulation of COX-2 expression during tumor progression. Importantly, down-regulation of COX-2 using small interfering RNA in cells showed no effect on cell proliferation, anchorage-independent growth, migration, or invasion. These results show that (a) COX-2 overexpression does not seem to predict a breast cancer precursor cell and does not provide advantage for the cell to be transformed; (b) inhibition of COX-2 does not affect hMEC growth and oncogenic behavior in the conditions analyzed; and (c) COX-2 expression is decreased in breast cancer cell lines and cancer specimens as compared with normal mammary epithelial cells.

Ada3 Requirement for HAT Recruitment to Estrogen Receptors and Estrogen-Dependent Breast Cancer Cell Proliferation

We have previously shown that evolutionarily conserved alteration/deficiency in activation (Ada) protein associates with and promotes estrogen receptor (ER)-mediated target gene expression. Here, we examined the role of endogenous Ada3 to recruit histone acetyl transferases (HAT) to an ER-responsive promoter and its role in estrogen-dependent cell proliferation and malignant phenotype. Using a combination of glycerol gradient cosedimentation and immunoprecipitation analyses, we show that Ada3, ER, and three distinct HATs [p300, (p300/CBP-associated factor) PCAF, and general control nonrepressed 5 (Gcn5)] are present in a complex. Using chromatin immunoprecipitation analysis, we show that short hairpin RNA (shRNA)-mediated knockdown of Ada3 in ER-positive breast cancer cells significantly reduced the ligand-dependent recruitment of p300, PCAF, and Gcn5 to the ER-responsive pS2 promoter. Finally, we use shRNA knockdown to show that Ada3 is critical for estrogen-dependent proliferation of ER-positive breast cancer cell lines in two-dimensional, as well as three-dimensional, culture. Knockdown of Ada3 in ER-positive MCF-7 cells induced reversion of the transformed phenotype in three-dimensional culture. Thus, our results show an important role of Ada3 in HAT recruitment to estrogen-responsive target gene promoters and for estrogen-dependent proliferation of breast cancer cells.

Overexpression of RhoA Induces Preneoplastic Transformation of Primary Mammary Epithelial Cells

Rho family small GTPases serve as molecular switches in the regulation of diverse cellular functions, including actin cytoskeleton remodeling, cell migration, gene transcription, and cell proliferation. Importantly, Rho overexpression is frequently seen in many carcinomas. However, published studies have almost invariably used immortal or tumorigenic cell lines to study Rho GTPase functions and there are no studies on the potential of Rho small GTPase to overcome senescence checkpoints and induce preneoplastic transformation of human mammary epithelial cells (hMEC). We show here that ectopic expression of wild-type (WT) RhoA as well as a constitutively active RhoA mutant (G14V) in two independent primary hMEC strains led to their immortalization and preneoplastic transformation. These cells have continued to grow over 300 population doublings (PD) with no signs of senescence, whereas cells expressing the vector or dominant-negative RhoA mutant (T19N) senesced after 20 PDs. Significantly, RhoA-T37A mutant, known to be incapable of interacting with many well-known Rho effectors including Rho kinase, PKN, mDia1, and mDia2, was also capable of immortalizing hMECs. Notably, similar to parental normal cells, Rho-immortalized cells have WT p53 and intact G(1) cell cycle arrest on Adriamycin treatment. Rho-immortalized cells were anchorage dependent and were unable to form tumors when implanted in nude mice. Lastly, microarray expression profiling of Rho-immortalized versus parental cells showed altered expression of several genes previously implicated in immortalization and breast cancer progression. Taken together, these results show that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer.

Distinct roles for Rho versus Rac/Cdc42 GTPases downstream of Vav2 in regulating mammary epithelial acinar architecture.

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.

The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells

BackgroundIdentification and characterization of molecular controls that regulate mammary stem and progenitor cell homeostasis are critical to our understanding of normal mammary gland development and its pathology.ResultsWe demonstrate that conditional knockout of Sox9 in the mouse mammary gland results in impaired postnatal development. In short-term lineage tracing in the postnatal mouse mammary gland using Sox9-CreER driven reporters, Sox9 marked primarily the luminal progenitors and bipotent stem/progenitor cells within the basal mammary epithelial compartment. In contrast, long-term lineage tracing studies demonstrate that Sox9+ precursors gave rise to both luminal and myoepithelial cell lineages. Finally, fate mapping of Sox9 deleted cells demonstrates that Sox9 is essential for luminal, but not myoepithelial, lineage commitment and proliferation.ConclusionsThese studies identify Sox9 as a key regulator of mammary gland development and stem/progenitor maintenance.

Overexpression of Ecdysoneless in Pancreatic Cancer and its Role in Oncogenesis by Regulating Glycolysis

Purpose: To study the expression and function of a novel cell-cycle regulatory protein, human ecdysoneless (Ecd), during pancreatic cancer pathogenesis. Experimental Design: Immunohistochemical expression profiling of Ecd was done in nonneoplastic normal pancreatic tissues and pancreatic ductal adenocarcinoma lesions (from tissue microarray and Rapid Autopsy program) as well as precancerous PanIN lesions and metastatic organs. To analyze the biological significance of Ecd in pancreatic cancer progression, Ecd was stably knocked down in pancreatic cancer cell line followed by in vitro and in vivo functional assays. Results: Normal pancreatic ducts showed very weak to no Ecd expression compared to significant positive expression in pancreatic cancer tissues (mean ± SE composite score: 0.3 ± 0.2 and 3.8 ± 0.2 respectively, P < 0.0001) as well as in PanIN precursor lesions with a progressive increase in Ecd expression with increasing dysplasia (PanIN-1–PanIN-3). Analysis of matched primary tumors and metastases from patients with pancreatic cancer revealed that Ecd is highly expressed in both primary pancreatic tumor and in distant metastatic sites. Furthermore, knockdown of Ecd suppressed cell proliferation in vitro and tumorigenicity of pancreatic cancer cells in mice orthotopic tumors. Microarray study revealed that Ecd regulates expression of glucose transporter GLUT4 in pancreatic cancer cells and was subsequently shown to modulate glucose uptake, lactate production, and ATP generation by pancreatic cancer cells. Finally, knockdown of Ecd also reduced level of pAkt, key signaling molecule known to regulate aerobic glycolysis in cancer cells. Conclusion: Ecd is a novel tumor-promoting factor that is differentially expressed in pancreatic cancer and potentially regulates glucose metabolism within cancer cells. Clin Cancer Res; 18(22); 6188–98. ©2012 AACR.