Lewis A. Chodosh

Differential Roles of Hypoxia-Inducible Factor 1α (HIF-1α) and HIF-2α in Hypoxic Gene Regulation

ABSTRACT Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF), a heterodimer of HIF-α and the aryl hydrocarbon receptor nuclear translocator subunits. The HIF-1α and HIF-2α subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes. Previous studies using Hif-1α−/− embryonic stem and mouse embryonic fibroblast cells show that loss of HIF-1α eliminates all oxygen-regulated transcriptional responses analyzed, suggesting that HIF-2α is dispensable for hypoxic gene regulation. In contrast, HIF-2α has been shown to regulate some hypoxia-inducible genes in transient transfection assays and during embryonic development in the lung and other tissues. To address this discrepancy, and to identify specific HIF-2α target genes, we used DNA microarray analysis to evaluate hypoxic gene induction in cells expressing HIF-2α but not HIF-1α. In addition, we engineered HEK293 cells to express stabilized forms of HIF-1α or HIF-2α via a tetracycline-regulated promoter. In this first comparative study of HIF-1α and HIF-2α target genes, we demonstrate that HIF-2α does regulate a variety of broadly expressed hypoxia-inducible genes, suggesting that its function is not restricted, as initially thought, to endothelial cell-specific gene expression. Importantly, HIF-1α (and not HIF-2α) stimulates glycolytic gene expression in both types of cells, clearly showing for the first time that HIF-1α and HIF-2α have unique targets.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozgyous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations

Although the process of mammary tumorigenesis requires multiple genetic events, it is unclear to what extent carcinogenesis proceeds through preferred secondary pathways following a specific initiating oncogenic event. Similarly, the extent to which established mammary tumors remain dependent on individual mutations for maintenance of the transformed state is unknown. Here we use the tetracycline regulatory system to conditionally express the human c-MYC oncogene in the mammary epithelium of transgenic mice. MYC encodes a transcription factor implicated in multiple human cancers. In particular, amplification and overexpression of c-MYC in human breast cancers is associated with poor prognosis, although the genetic mechanisms by which c-MYC promotes tumor progression are poorly understood. We show that deregulated c-MYC expression in this inducible system results in the formation of invasive mammary adenocarcinomas, many of which fully regress following c-MYC deinduction. Approximately half of these tumors harbor spontaneous activating point mutations in the ras family of proto-oncogenes with a strong preference for Kras2 compared with Hras1. Nearly all tumors lacking activating ras mutations fully regressed following c-MYC deinduction, whereas tumors bearing ras mutations did not, suggesting that secondary mutations in ras contribute to tumor progression. These findings demonstrate that c-MYC-induced mammary tumorigenesis proceeds through a preferred secondary oncogenic pathway involving Kras2.

The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues.

We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17β–estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones.

Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis

Activating Ras mutations can induce either proliferation or senescence depending on the cellular context. To determine whether Ras activation has context-dependent effects in the mammary gland, we generated doxycycline-inducible transgenic mice that permit Ras activation to be titrated. Low levels of Ras activation — similar to those found in non-transformed mouse tissues expressing endogenous oncogenic Kras2 — stimulate cellular proliferation and mammary epithelial hyperplasias. In contrast, high levels of Ras activation — similar to those found in tumours bearing endogenous Kras2 mutations — induce cellular senescence that is Ink4a–Arf- dependent and irreversible following Ras downregulation. Chronic low-level Ras induction results in tumour formation, but only after the spontaneous upregulation of activated Ras and evasion of senescence checkpoints. Thus, high-level, but not low-level, Ras activation activates tumour suppressor pathways and triggers an irreversible senescent growth arrest in vivo. We suggest a three-stage model for Ras-induced tumorigenesis consisting of an initial activating Ras mutation, overexpression of the activated Ras allele and, finally, evasion of p53–Ink4a–Arf-dependent senescence checkpoints.

CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression

Epithelial-mesenchymal transition (EMT) is a tightly regulated process that is critical for embryogenesis but is abnormally activated during cancer metastasis and recurrence. Here we show that a switch in CD44 alternative splicing is required for EMT. Using both in vitro and in vivo systems, we have demonstrated a shift in CD44 expression from variant isoforms (CD44v) to the standard isoform (CD44s) during EMT. This isoform switch to CD44s was essential for cells to undergo EMT and was required for the formation of breast tumors that display EMT characteristics in mice. Mechanistically, the splicing factor epithelial splicing regulatory protein 1 (ESRP1) controlled the CD44 isoform switch and was critical for regulating the EMT phenotype. Additionally, the CD44s isoform activated Akt signaling, providing a mechanistic link to a key pathway that drives EMT. Finally, CD44s expression was upregulated in high-grade human breast tumors and was correlated with the level of the mesenchymal marker N-cadherin in these tumors. Together, our data suggest that regulation of CD44 alternative splicing causally contributes to EMT and breast cancer progression.

Hlx is induced by and genetically interacts with T-bet to promote heritable T H 1 gene induction

Type 1 helper T (TH1) cells are essential for cellular immunity, but their ontogeny, maturation and durability remain poorly understood. By constructing a dominant-negative form of T-bet, we were able to determine the role played by this lineage-inducing trans-activator in the establishment and maintenance of heritable TH1 gene expression. Optimal induction of interferon-γ (IFN-γ) expression required genetic interaction between T-bet and its target, the homeoprotein Hlx. In fully mature TH1 cells, reiteration of IFN-γ expression and stable chromatin remodeling became relatively independent of T-bet activity and coincided with demethylation of DNA. In contrast, some lineage attributes, such as expression of IL-12Rβ2 (interleukin 12 receptor β2), required ongoing T-bet activity in mature TH1 cells and their progeny. These findings suggest that heritable states of gene expression might be maintained by continued expression of the inducing factor or by a mechanism that confers a stable imprint of the induced state.

Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis

To determine the impact of tumor progression on the reversibility of Neu-induced tumorigenesis, we have used the tetracycline regulatory system to conditionally express activated Neu in the mammary epithelium of transgenic mice. When induced with doxycycline, bitransgenic MMTV-rtTA/TetO-NeuNT mice develop multiple invasive mammary carcinomas, essentially all of which regress to a clinically undetectable state following transgene deinduction. This demonstrates that Neu-initiated tumorigenesis is reversible. Strikingly, extensive lung metastases arising from Neu-induced mammary tumors also rapidly and fully regress following the abrogation of Neu expression. However, despite the near universal dependence of both primary tumors and metastases on Neu transgene expression, most animals bearing fully regressed Neu-induced tumors ultimately develop recurrent tumors that have progressed to a Neu-independent state.

A targeted proteomics–based pipeline for verification of biomarkers in plasma

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.

mTOR Mediates Wnt-Induced Epidermal Stem Cell Exhaustion and Aging

Epidermal integrity is a complex process established during embryogenesis and maintained throughout the organism lifespan by epithelial stem cells. Although Wnt regulates normal epithelial stem cell renewal, aberrant Wnt signaling can contribute to cancerous growth. Here, we explored the consequences of persistent expressing Wnt1 in an epidermal compartment that includes the epithelial stem cells. Surprisingly, Wnt caused the rapid growth of the hair follicles, but this was followed by epithelial cell senescence, disappearance of the epidermal stem cell compartment, and progressive hair loss. Although Wnt1 induced the activation of beta-catenin and the mTOR pathway, both hair follicle hyperproliferation and stem cell exhaustion were strictly dependent on mTOR function. These findings suggest that whereas activation of beta-catenin contributes to tumor growth, epithelial stem cells may be endowed with a protective mechanism that results in cell senescence upon the persistent stimulation of proliferative pathways that activate mTOR, ultimately suppressing tumor formation.