Summary

Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression and regression. Mass spectrometry data derived from ∼ 1.6 million spectra identified protein networks involved in wound healing, microenvironment and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth, and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the non-cancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a non-specific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiological processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development with relevance to plasma-based strategies to detect and diagnose cancer.

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed “intrinsic tumor suppressor activity,” and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.

Purpose

We generated extensive transcriptional and proteomic profiles from a Her2-driven mouse model of breast cancer that closely recapitulates human breast cancer. This report makes these data publicly available in raw and processed forms, as a resource to the community. Importantly, we previously made biospecimens from this same mouse model freely available through a sample repository, so researchers can obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens.

Experimental design

Twelve datasets are available, encompassing 841 LC-MS/MS experiments (plasma and tissues) and 255 microarray analyses of multiple tissues (thymus, spleen, liver, blood cells, and breast). Cases and controls were rigorously paired to avoid bias.

Results

In total, 18,880 unique peptides were identified (PeptideProphet peptide error rate ≤1%), with 3884 and 1659 non-redundant protein groups identified in plasma and tissue datasets, respectively. Sixty-one of these protein groups overlapped between cancer plasma and cancer tissue.

Conclusions and clinical relevance

These data are of use for advancing our understanding of cancer biology, for software and quality control tool development, investigations of analytical variation in MS/MS data, and selection of proteotypic peptides for MRM-MS. The availability of these datasets will contribute positively to clinical proteomics.

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.

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development.

We developed a doxycycline-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with doxycycline from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared to PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing CyclinD1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared to vehicle-treated control. This data indicates that epithelial NF-κB is an active contributor to tumor progression and demonstrates that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that functions as an oncogene or tumor suppressor in a highly tissue-specific cell-dependent manner. However, its precise role in breast cancer and metastasis remains unclear. Here, we show that transient adenoviral expression of KLF4 in the 4T1 orthotopic mammary cancer model significantly attenuated primary tumor growth as well as micrometastases to the lungs and liver. These results can be attributed, in part, to decreased proliferation and increased apoptosis. Further supporting a tumor-suppressive role for KLF4 in the breast, we found that KLF4 expression is lost in a mouse model of HER2/NEU/ERBB2-positive breast cancer. To determine whether enforced KLF4 expression could alter tumor latency in these mice, we used a doxycycline-inducible expression model in the context of the MMTV-Neu transgene. Surprisingly, tumors that developed in this model also lost KLF4 expression, suggesting negative selection for sustained expression. We have previously reported that KLF4 inhibits epithelial-to-mesenchymal transition (EMT), a preliminary step in metastatic progression. Overexpression of KLF4 in 4T1 cells led to a significant reduction in the expression of Snail, a key mediator of EMT and metastasis. Conversely, KLF4 silencing increased Snail expression in the nontransformed MCF-10A cell line. Collectively, these data demonstrate the first functional, in vivo evidence for KLF4 as a tumor suppressor in breast cancer cells. Furthermore, our findings suggest an inhibitory role for KLF4 during breast cancer metastases that functions, in part, through repression of Snail.

Understanding the molecular pathways that contribute to the aggressive behavior of human cancers is a critical research priority. The SNF1/AMPK-related protein kinase Hunk is overexpressed in aggressive subsets of human breast, ovarian, and colon cancers. Analysis of Hunk–/– mice revealed that this kinase is required for metastasis of c-myc–induced mammary tumors but not c-myc–induced primary tumor formation. Similar to c-myc, amplification of the proto-oncogene HER2/neu occurs in 10%–30% of breast cancers and is associated with aggressive tumor behavior. By crossing Hunk–/– mice with transgenic mouse models for HER2/neu-induced mammary tumorigenesis, we report that Hunk is required for primary tumor formation induced by HER2/neu. Knockdown and reconstitution experiments in mouse and human breast cancer cell lines demonstrated that Hunk is required for maintenance of the tumorigenic phenotype in HER2/neu-transformed cells. This requirement is kinase dependent and resulted from the ability of Hunk to suppress apoptosis in association with downregulation of the tumor suppressor p27kip1. Additionally, we find that Hunk is rapidly upregulated following HER2/neu activation in vivo and in vitro. These findings provide what we believe is the first evidence for a role for Hunk in primary tumorigenesis and cell survival and identify this kinase as an essential effector of the HER2/neu oncogenic pathway.

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.

We investigated whether Nuclear Factor-kappa B (NF-κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and remodeling. We generated a doxycycline inducible transgenic mouse model to activate NF-κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF-κB signaling cascade. We found that activation of NF-κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase-3, an indicator of apoptosis. We also found that activation of NF-κB during lactation with no additional involution signals had a similar effect. The observation that NF-κB is a key regulator of milk production led us to investigate the role of NF-κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF-κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF-κB in mammary epithelium. Together, our data reveal that activation of NF-κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF-κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact.

Parathyroid hormone-related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP’s effects on mesenchymal cells, we profiled gene expression in WT and PTHrP−/− mammary buds, and in WT and K14-PTHrP ventral skin at E15.5. By cross-referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds.

Introduction

Rho GTPases are overexpressed and hyperactivated in human breast cancers. Deficiency of p190B RhoGAP, a major inhibitor of the Rho GTPases, inhibits mouse mammary tumor virus long terminal repeat (MMTV)-Neu/ErbB2 mammary tumor formation and progression in part through effects within the stromal environment, suggesting that p190B function is pro-tumorigenic. To further investigate the potential pro-tumorigenic actions of p190B, we examined the effects of exogenous p190B expression within the mammary epithelium on MMTV-Neu tumor formation and progression.

Methods

Tetracycline (tet)-regulatable p190B transgenic mice were bred to MMTV-Neu mice, and the effects of exogenous p190B expression on tumor latency, multiplicity, growth rates, angiogenesis, and metastasis were examined. The effects of exogenous p190B expression on cell-matrix adhesion and invasion were tested using non-transformed primary mammary epithelial cells (MECs). Rho GTPase activity, oxidative stress as an indicator of reactive oxygen species (ROS) production, and downstream signaling pathways were analyzed.

Results

Altered p190B expression resulted in a two-fold increase in tumor multiplicity and a three-fold increase in metastases compared to control mice indicating that exogenous p190B expression in the mammary epithelium promotes MMTV-Neu mammary tumor formation and progression. Interestingly, non-transformed primary MECs expressing exogenous p190B displayed increased adhesion to laminin and type IV collagen and formed invasive structures in a three-dimensional culture assay. Ras related C3 botulinum toxin 1 (Rac1)-GTP levels were elevated in p190B transgenic tumors whereas Ras homologous A (RhoA) and cell division cycle 42 (Cdc42)-GTP levels were not significantly altered. Rac1 activity affects production of ROS, which regulate transformation, metastasis, and oxidative stress. Protein carbonylation, which is indicative of oxidative stress, was elevated 1.75-fold in p190B transgenic tumors as compared to control tumors suggesting that exogenous p190B expression may affect Rac1-dependent ROS production.

Conclusions

These studies indicate that paradoxically, p190B RhoGAP, a major inhibitor of the Rho GTPases in vitro, has pro-tumorigenic functions that enhance MMTV-Neu induced mammary tumor formation and metastasis. Furthermore, exogenous p190B expression enhances cell adhesion and invasion, which may facilitate metastasis. Rac1 activity and oxidative stress are elevated in tumors expressing exogenous p190B suggesting that p190B may promote tumorigenesis through a Rac1/ROS dependent mechanism.

Introduction

The Akt pathway plays a central role in regulating cell survival, proliferation and metabolism, and is one of the most commonly activated pathways in human cancer. A role for Akt in epithelial differentiation, however, has not been established. We previously reported that mice lacking Akt1, but not Akt2, exhibit a pronounced metabolic defect during late pregnancy and lactation that results from a failure to upregulate Glut1 as well as several lipid synthetic enzymes. Despite this metabolic defect, however, both Akt1-deficient and Akt2-deficient mice exhibit normal mammary epithelial differentiation and Stat5 activation.

Methods

In light of the overlapping functions of Akt family members, we considered the possibility that Akt may play an essential role in regulating mammary epithelial development that is not evident in Akt1-deficient mice due to compensation by other Akt isoforms. To address this possibility, we interbred mice bearing targeted deletions in Akt1 and Akt2 and determined the effect on mammary differentiation during pregnancy and lactation.

Results

Deletion of one allele of Akt2 in Akt1-deficient mice resulted in a severe defect in Stat5 activation during late pregnancy that was accompanied by a global failure of terminal mammary epithelial cell differentiation, as manifested by the near-complete loss in production of the three principal components of milk: lactose, lipid, and milk proteins. This defect was due, in part, to a failure of pregnant Akt1-/-;Akt2+/- mice to upregulate the positive regulator of Prlr-Jak-Stat5 signaling, Id2, or to downregulate the negative regulators of Prlr-Jak-Stat5 signaling, caveolin-1 and Socs2.

Conclusions

Our findings demonstrate an unexpected requirement for Akt in Prlr-Jak-Stat5 signaling and establish Akt as an essential central regulator of mammary epithelial differentiation and lactation.

Data concerning the prognostic value of ErbB4 in breast cancer and effects on cell growth have varied in published reports, perhaps due to the unknown signaling consequences of expression of the intracellular proteolytic ErbB4 s80HER4 fragment or due to differing signaling capabilities of alternatively spliced ErbB4 isoforms. One isoform (Cyt1) contains a 16-residue intracellular sequence that is absent from the other (Cyt2). We expressed s80Cyt1 and s80Cyt2 in HC11 mammary epithelial cells, finding diametrically opposed effects on the growth and organization of colonies in three-dimensional matrices. Whereas expression of s80Cyt1 decreased growth and increased the rate of three-dimensional lumen formation, that of s80Cyt2 increased proliferation without promoting lumen formation. These results were recapitulated in vivo, using doxycycline-inducible, mouse breast-transgenic expression of s80Cyt1 amd s80Cyt2. Expression of s80Cyt1 decreased growth of the mammary ductal epithelium, caused precocious STAT5a activation and lactogenic differentiation, and increased cell surface E-cadherin levels. Remarkably, ductal growth inhibition by s80Cyt1 occurred simultaneously with lobuloalveolar growth that was unimpeded by s80Cyt1, suggesting that the response to ErbB4 may be influenced by the epithelial subtype. In contrast, expression of s80Cyt2 caused epithelial hyperplasia, increased Wnt and nuclear β-catenin expression, and elevated expression of c-myc and cyclin D1 in the mammary epithelium. These results demonstrate that the Cyt1 and Cyt2 ErbB4 isoforms, differing by only 16 amino acids, exhibit markedly opposing effects on mammary epithelium growth and differentiation.

Six1 is a developmentally regulated homeoprotein with limited expression in most normal adult tissues and frequent misexpression in a variety of malignancies. Here we demonstrate, using a bitransgenic mouse model, that misexpression of human Six1 in adult mouse mammary gland epithelium induces tumors of multiple histological subtypes in a dose-dependent manner. The neoplastic lesions induced by Six1 had an in situ origin, showed diverse differentiation, and exhibited progression to aggressive malignant neoplasms, as is often observed in human carcinoma of the breast. Strikingly, the vast majority of Six1-induced tumors underwent an epithelial-mesenchymal transition (EMT) and expressed multiple targets of activated Wnt signaling, including cyclin D1. Interestingly, Six1 and cyclin D1 coexpression was found to frequently occur in human breast cancers and was strongly predictive of poor prognosis. We further show that Six1 promoted a stem/progenitor cell phenotype in the mouse mammary gland and in Six1-driven mammary tumors. Our data thus provide genetic evidence for a potent oncogenic role for Six1 in mammary epithelial neoplasia, including promotion of EMT and stem cell–like features.

The oncogene erbB2 is overexpressed in 20% to 30% human breast cancers and is most commonly overexpressed in estrogen receptor (ER)-negative breast cancers. Transgenic mice expressing erbB2 develop ER-negative mammary tumors, mimicking human breast carcinogenesis. Previously, we have shown that activator protein 1 (AP-1) regulates proliferation of ER-negative breast cancer cells. We hypothesized that blockade of AP-1 in mouse mammary epithelial cells will suppress ER-negative tumorigenesis induced by erbB2. Trigenic erbB2 mice were generated by crossing a bigenic pUHD-Tam67/MMTV-rtTA mouse to a MMTV-erbB2 mouse. The resulting trigenic mice develop tumors and express a doxycycline-inducible c-Jun dominant negative mutant (Tam67) in the mammary glands. In vivo AP-1 blockade by Tam67 expression delayed mammary tumor formation in MMTV-erbB2 mice by more than 11 weeks. By 52 weeks of age, 100% (18 of 18) of the untreated animals had developed mammary tumors, whereas 56% (9 of 16) of the doxycycline-treated trigenic mice developed tumors. In addition, the tumors that arose in the AP-1-blocked erbB2 mice failed to express Tam67. Twenty-five percent of the doxycycline-treated MMTV-erbB2 mice survived more than 72 weeks of age without developing mammary tumors. Examination of normal-appearing mammary glands from these mice showed that AP-1 blockade by Tam67 also significantly prevents the development of premalignant lesions in these glands. The expression of erbB2 either in normal mammary tissue or in mammary tumors was not altered. Our results show that blocking the AP-1 signaling in mammary cells suppresses erbB2-induced transformation, and show that the AP-1 transcription factor is a critical transducer of erbB2. These results provide a scientific rationale to develop targeted drugs that inhibit AP-1 to prevent the development of ER-negative breast cancer.

Singular value decomposition regression can detect the activation of endogenous signaling pathways, allowing the identification of pathway cross-talk.

The ability to detect activation of signaling pathways based solely on gene expression data represents an important goal in biological research. We tested the sensitivity of singular value decomposition-based regression by focusing on functional interactions between the Ras and transforming growth factor beta signaling pathways. Our findings demonstrate that this approach is sufficiently sensitive to detect the secondary activation of endogenous signaling pathways as it occurs through crosstalk following ectopic activation of a primary pathway.

Breast cancers frequently progress or relapse during targeted therapy, but the molecular mechanisms that enable escape remain poorly understood. We elucidated genetic determinants underlying tumor escape in a transgenic mouse model of Wnt pathway–driven breast cancer, wherein targeted therapy is simulated by abrogating doxycycline-dependent Wnt1 transgene expression within established tumors. In mice with intact tumor suppressor pathways, tumors typically circumvented doxycycline withdrawal by reactivating Wnt signaling, either via aberrant (doxycycline-independent) Wnt1 transgene expression or via acquired somatic mutations in the gene encoding β-catenin. Germline introduction of mutant tumor suppressor alleles into the model altered the timing and mode of tumor escape. Relapses occurring in the context of null Ink4a/Arf alleles (disrupting both the p16Ink4a and p19Arf tumor suppressors) arose quickly and rarely reactivated the Wnt pathway. In addition, Ink4a/Arf-deficient relapses resembled p53-deficient relapses in that both displayed morphologic and molecular hallmarks of an epithelial-to-mesenchymal transition (EMT). Notably, Ink4a/Arf deficiency promoted relapse in the absence of gross genomic instability. Moreover, Ink4a/Arf-encoded proteins differed in their capacity to suppress oncogene independence. Isolated p19Arf deficiency mirrored p53 deficiency in that both promoted rapid, EMT-associated mammary tumor escape, whereas isolated p16Ink4a deficiency failed to accelerate relapse. Thus, p19Arf/p53 pathway lesions may promote mammary cancer relapse even when inhibition of a targeted oncogenic signaling pathway remains in force.

c-myc is frequently amplified in breast cancer; however, the mechanism of myc-induced mammary epithelial cell transformation has not been defined. We show that c-Myc induces a profound morphological transformation in human mammary epithelial cells and anchorage-independent growth. c-Myc suppresses the Wnt inhibitors DKK1 and SFRP1, and derepression of DKK1 or SFRP1 reduces Myc-dependent transforming activity. Myc-dependent repression of DKK1 and SFRP1 is accompanied by Wnt target gene activation and endogenous T-cell factor activity. Myc-induced mouse mammary tumors have repressed SFRP1 and increased expression of Wnt target genes. DKK1 and SFRP1 inhibit the transformed phenotype of breast cancer cell lines, and DKK1 inhibits tumor formation. We propose a positive feedback loop for activation of the c-myc and Wnt pathways in breast cancer.

We have previously shown that c-MYC-induced mammary tumorigenesis in mice proceeds via a preferred secondary pathway involving spontaneous activating mutations in Kras2 (C. M. D'Cruz, E. J. Gunther, R. B. Boxer, J. L. Hartman, L. Sintasath, S. E. Moody, J. D. Cox, S. I. Ha, G. K. Belka, A. Golant, R. D. Cardiff, and L. A. Chodosh, Nat. Med. 7:235-239, 2001). In contrast, we now demonstrate that Wnt1-induced mammary tumorigenesis proceeds via a pathway that preferentially activates Hras1. In addition, we find that expression of oncogenic forms of Kras2 and Hras1 from their endogenous promoters has markedly different consequences for the progression of tumors to oncogene independence. Spontaneous activating Kras2 mutations occurring in either MYC- or Wnt1-induced tumors were strongly associated with oncogene-independent tumor growth following MYC or Wnt1 downregulation. In contrast, Hras1-mutant Wnt1-induced tumors consistently remained oncogene dependent. Additionally, Kras2-mutant tumors exhibited substantially higher levels of ras-GTP, phospho-Erk1/2, and phospho-Mek1/2 compared to Hras1-mutant tumors, suggesting the involvement of the ras/mitogen-activated protein kinase (MAPK) pathway in the acquisition of oncogene independence. Consistent with this, by use of carcinogen-induced ras mutations as well as knock-in mice harboring a latent activated Kras2 allele, we demonstrate that Kras2 activation strongly synergizes with both c-MYC and Wnt1 in mammary tumorigenesis and promotes the progression of tumors to oncogene independence. Together, our findings support a model for tumorigenesis in which c-MYC and Wnt1 select for the outgrowth of cells harboring mutations in specific ras isoforms and that these secondary mutations, in turn, determine the extent of ras/MAPK pathway activation and the potential for oncogene-independent growth.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α. 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 and require distinct transcriptional cofactors. Our previous results demonstrated that HIF-1α and HIF-2α regulate distinct target genes. Here, we report that HIF-2α is not transcriptionally active in embryonic stem (ES) cells, as well as possible inhibition by a HIF-2α-specific transcriptional repressor. Using DNA microarray analysis of hypoxia-inducible genes in wild-type (WT), Hif-1α−/−, and Hif-2α−/− ES cells, we show that HIF-1α induces a large number of both confirmed and novel hypoxia-inducible genes, while HIF-2α does not activate any of its previously described targets. We further demonstrate that inhibition of HIF-2α function occurs at the level of transcription cofactor recruitment to endogenous target gene promoters. Overexpression of WT and, notably, a DNA-binding-defective HIF-2α mutant restores endogenous HIF-2α protein activity, suggesting that ES cells express a HIF-2α-specific corepressor that can be titrated by overexpressed HIF-2α protein. HIF-2α repression may explain why patients with mutations in the VHL tumor suppressor gene display cancerous lesions in specific tissue types.

Background

Microarray expression profiling has been widely used to identify differentially expressed genes in complex cellular systems. However, while such methods can be used to directly infer intracellular regulation within homogeneous cell populations, interpretation of in vivo gene expression data derived from complex organs composed of multiple cell types is more problematic. Specifically, observed changes in gene expression may be due either to changes in gene regulation within a given cell type or to changes in the relative abundance of expressing cell types. Consequently, bona fide changes in intrinsic gene regulation may be either mimicked or masked by changes in the relative proportion of different cell types. To date, few analytical approaches have addressed this problem.

Results

We have chosen to apply a computational method for deconvoluting gene expression profiles derived from intact tissues by using reference expression data for purified populations of the constituent cell types of the mammary gland. These data were used to estimate changes in the relative proportions of different cell types during murine mammary gland development and Ras-induced mammary tumorigenesis. These computational estimates of changing compartment sizes were then used to enrich lists of differentially expressed genes for transcripts that change as a function of intrinsic intracellular regulation rather than shifts in the relative abundance of expressing cell types. Using this approach, we have demonstrated that adjusting mammary gene expression profiles for changes in three principal compartments – epithelium, white adipose tissue, and brown adipose tissue – is sufficient both to reduce false-positive changes in gene expression due solely to changes in compartment sizes and to reduce false-negative changes by unmasking genuine alterations in gene expression that were otherwise obscured by changes in compartment sizes.

Conclusion

By adjusting gene expression values for changes in the sizes of cell type-specific compartments, this computational deconvolution method has the potential to increase both the sensitivity and specificity of differential gene expression experiments performed on complex tissues. Given the necessity for understanding complex biological processes such as development and carcinogenesis within the context of intact tissues, this approach offers substantial utility and should be broadly applicable to identifying gene expression changes in tissues composed of multiple cell types.

A novel algorithm (ChipStat) is presented for detecting gene-expression changes from Affymetrix microarray data. The method is used to identify changes in murine mammary development.

We describe a novel algorithm (ChipStat) for detecting gene-expression changes utilizing probe-level comparisons of replicate Affymetrix oligonucleotide microarray data. A combined detection approach is shown to yield greater sensitivity than a number of widely used methodologies including SAM, dChip and logit-T. Using this approach, we identify alterations in functional pathways during murine neonatal-pubertal mammary development that include the coordinate upregulation of major urinary proteins and the downregulation of loci exhibiting reciprocal imprinting.

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene targeting. Brca1S971A/S971A mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1S971A/S971A mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1S971A/S971A mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1S971A/S971A cells displayed reduced ability to activate the G2/M cell cycle checkpoint upon γ-irradiation and to stabilize p53 following N-methyl-N′-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.

The developmental preparation of the mammary gland for milk production that occurs during pregnancy is followed by an equally dramatic process of involution as the gland returns to its prepregnancy state. Two detailed, microarray-based surveys reported in this issue extend our understanding of the nature and timing of molecular and cellular events in involution that underlie these developmental changes.