The Evolving Landscape of B Cells in Cancer Metastasis.

Metastasis is the leading cause of cancer mortality. Functional and clinical studies have documented diverse B-cell and antibody responses in cancer metastasis. The presence of B cells in tumor microenvironments and metastatic sites has been associated with diverse effects that can promote or inhibit metastasis. Specifically, B cells can contribute to the spread of cancer cells by enhancing tumor cell motility, invasion, angiogenesis, lymphangiogenesis, and extracellular matrix remodeling. Moreover, they can promote metastatic colonization by triggering pathogenic immunoglobulin responses and recruiting immune suppressive cells. Contrastingly, B cells can also exhibit antimetastatic effects. For example, they aid in enhanced antigen presentation, which helps activate immune responses against cancer cells. In addition, B cells play a crucial role in preventing the dissemination of metastatic cells from the primary tumor and secrete antibodies that can aid in tumor recognition. Here, we review the complex roles of B cells in metastasis, delineating the heterogeneity of B-cell activity and subtypes by metastatic site, antibody class, antigen (if known), and molecular phenotype. These important attributes of B cells emphasize the need for a deeper understanding and characterization of B-cell phenotypes to define their effects in metastasis.

Elevated phosphorylation of EGFR in NSCLC due to mutations in PTPRH.

The role of EGFR in lung cancer is well described with numerous activating mutations that result in phosphorylation and tyrosine kinase inhibitors that target EGFR. While the role of the EGFR kinase in non-small cell lung cancer (NSCLC) is appreciated, control of EGFR signaling pathways through dephosphorylation by phosphatases is not as clear. Through whole genome sequencing we have uncovered conserved V483M Ptprh mutations in PyMT induced tumors. Profiling the downstream events of Ptprh mutant tumors revealed AKT activation, suggesting a key target of PTPRH was EGFR tyrosine 1197. Given the role of EGFR in lung cancer, we explored TCGA data which revealed that a subset of PTPRH mutant tumors shared gene expression profiles with EGFR mutant tumors, but that EGFR mutations and PTPRH mutations were mutually exclusive. Generation of a PTPRH knockout NSCLC cell line resulted in Y1197 phosphorylation of EGFR, and a rescue with expression of wild type PTPRH returned EGFR phosphorylation to parental line values while rescue with catalytically dead PTPRH did not. A dose response curve illustrated that two human NSCLC lines with naturally occurring PTPRH mutations responded to EGFR tyrosine kinase inhibition. Osimertinib treatment of these tumors resulted in a reduction of tumor volume relative to vehicle controls. PTPRH mutation resulted in nuclear pEGFR as seen in immunohistochemistry, suggesting that there may also be a role for EGFR as a transcriptional co-factor. Together these data suggest mutations in PTPRH in NSCLC is inhibitory to PTPRH function, resulting in aberrant EGFR activity and ultimately may result in clinically actionable alterations using existing therapies.

Tumour-infiltrating B cells: immunological mechanisms, clinical impact and therapeutic opportunities.

Although immunotherapy research to date has focused largely on T cells, there is mounting evidence that tumour-infiltrating B cells and plasma cells (collectively referred to as tumour-infiltrating B lymphocytes (TIL-Bs)) have a crucial, synergistic role in tumour control. In many cancers, TIL-Bs have demonstrated strong predictive and prognostic significance in the context of both standard treatments and immune checkpoint blockade, offering the prospect of new therapeutic opportunities that leverage their unique immunological properties. Drawing insights from autoimmunity, we review the molecular phenotypes, architectural contexts, antigen specificities, effector mechanisms and regulatory pathways relevant to TIL-Bs in human cancer. Although the field is young, the emerging picture is that TIL-Bs promote antitumour immunity through their unique mode of antigen presentation to T cells; their role in assembling and perpetuating immunologically 'hot' tumour microenvironments involving T cells, myeloid cells and natural killer cells; and their potential to combat immune editing and tumour heterogeneity through the easing of self-tolerance mechanisms. We end by discussing the most promising approaches to enhance TIL-B responses in concert with other immune cell subsets to extend the reach, potency and durability of cancer immunotherapy.

Microneedle-mediated Intratumoral Delivery of Anti-CTLA-4 Promotes cDC1-dependent Eradication of Oral Squamous Cell Carcinoma with Limited irAEs.

Head and neck squamous cell carcinoma (HNSCC) ranks sixth in cancer incidence worldwide and has a 5-year survival rate of only 63%. Immunotherapies-principally immune checkpoint inhibitors (ICI), such as anti-PD-1 and anti-CTLA-4 antibodies that restore endogenous antitumor T-cell immunity-offer the greatest promise for HNSCC treatment. Anti-PD-1 has been recently approved for first-line treatment of recurrent and metastatic HNSCC; however, less than 20% of patients show clinical benefit and durable responses. In addition, the clinical application of ICI has been limited by immune-related adverse events (irAE) consequent to compromised peripheral immune tolerance. Although irAEs are often reversible, they can become severe, prompting premature therapy termination or becoming life threatening. To address the irAEs inherent to systemic ICI therapy, we developed a novel, local delivery strategy based upon an array of soluble microneedles (MN). Using our recently reported syngeneic, tobacco-signature murine HNSCC model, we found that both systemic and local-MN anti-CTLA-4 therapy lead to >90% tumor response, which is dependent on CD8 T cells and conventional dendritic cell type 1 (cDC1). However, local-MN delivery limited the distribution of anti-CTLA-4 antibody from areas distal to draining lymphatic basins. Employing Foxp3-GFPDTR transgenic mice to interrogate irAEs in vivo, we found that local-MN delivery of anti-CTLA-4 protects animals from irAEs observed with systemic therapy. Taken together, our findings support the exploration of MN-intratumoral ICI delivery as a viable strategy for HNSCC treatment with reduced irAEs, and the opportunity to target cDC1s as part of multimodal treatment options to boost ICI therapy.

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

FOXA1 overexpression suppresses interferon signaling and immune response in cancer.

Androgen receptor-positive prostate cancer (PCa) and estrogen receptor-positive luminal breast cancer (BCa) are generally less responsive to immunotherapy compared with certain tumor types such as melanoma. However, the underlying mechanisms are not fully elucidated. In this study, we found that FOXA1 overexpression inversely correlated with interferon (IFN) signature and antigen presentation gene expression in PCa and BCa patients. FOXA1 bound the STAT2 DNA-binding domain and suppressed STAT2 DNA-binding activity, IFN signaling gene expression, and cancer immune response independently of the transactivation activity of FOXA1 and its mutations detected in PCa and BCa. Increased FOXA1 expression promoted cancer immuno- and chemotherapy resistance in mice and PCa and BCa patients. These findings were also validated in bladder cancer expressing high levels of FOXA1. FOXA1 overexpression could be a prognostic factor to predict therapy resistance and a viable target to sensitize luminal PCa, BCa, and bladder cancer to immuno- and chemotherapy.

PDGFRß is an essential therapeutic target for BRCA1-deficient mammary tumors.

BACKGROUND: Basal-like breast cancers (BLBCs) are a leading cause of cancer death due to their capacity to metastasize and lack of effective therapies. More than half of BLBCs have a dysfunctional BRCA1. Although most BRCA1-deficient cancers respond to DNA-damaging agents, resistance and tumor recurrence remain a challenge to survival outcomes for BLBC patients. Additional therapies targeting the pathways aberrantly activated by BRCA1 deficiency are urgently needed. METHODS: Most BRCA1-deficient BLBCs carry a dysfunctional INK4-RB pathway. Thus, we created genetically engineered mice with Brca1 loss and deletion of p16INK4A, or separately p18INK4C, to model the deficient INK4-RB signaling in human BLBC. By using these mutant mice and human BRCA1-deficient and proficient breast cancer tissues and cells, we tested if there exists a druggable target in BRCA1-deficient breast cancers. RESULTS: Heterozygous germline or epithelium-specific deletion of Brca1 in p18INK4C- or p16INK4A-deficient mice activated Pdgfrß signaling, induced epithelial-to-mesenchymal transition, and led to BLBCs. Confirming this role, targeted deletion of Pdgfrß in Brca1-deficient tumor cells promoted cell death, induced mesenchymal-to-epithelial transition, and suppressed tumorigenesis. Importantly, we also found that pharmaceutical inhibition of Pdgfrß and its downstream target Pkcα suppressed Brca1-deficient tumor initiation and progression and effectively killed BRCA1-deficient cancer cells. CONCLUSIONS: Our work offers the first genetic and biochemical evidence that PDGFRß-PKCα signaling is repressed by BRCA1, which establishes PDGFRß-PKCα signaling as a therapeutic target for BRCA1-deficient breast cancers.

Stimulation of Oncogene-Specific Tumor-Infiltrating T Cells through Combined Vaccine and αPD-1 Enable Sustained Antitumor Responses against Established HER2 Breast Cancer.

PURPOSE: Despite promising advances in breast cancer immunotherapy, augmenting T-cell infiltration has remained a significant challenge. Although neither individual vaccines nor immune checkpoint blockade (ICB) have had broad success as monotherapies, we hypothesized that targeted vaccination against an oncogenic driver in combination with ICB could direct and enable antitumor immunity in advanced cancers. EXPERIMENTAL DESIGN: Our models of HER2+ breast cancer exhibit molecular signatures that are reflective of advanced human HER2+ breast cancer, with a small numbers of neoepitopes and elevated immunosuppressive markers. Using these, we vaccinated against the oncogenic HER2Δ16 isoform, a nondriver tumor-associated gene (GFP), and specific neoepitopes. We further tested the effect of vaccination or anti-PD-1, alone and in combination. RESULTS: We found that only vaccination targeting HER2Δ16, a driver of oncogenicity and HER2-therapeutic resistance, could elicit significant antitumor responses, while vaccines targeting a nondriver tumor-specific antigen or tumor neoepitopes did not. Vaccine-induced HER2-specific CD8+ T cells were essential for responses, which were more effective early in tumor development. Long-term tumor control of advanced cancers occurred only when HER2Δ16 vaccination was combined with αPD-1. Single-cell RNA sequencing of tumor-infiltrating T cells revealed that while vaccination expanded CD8 T cells, only the combination of vaccine with αPD-1 induced functional gene expression signatures in those CD8 T cells. Furthermore, we show that expanded clones are HER2-reactive, conclusively demonstrating the efficacy of this vaccination strategy in targeting HER2. CONCLUSIONS: Combining oncogenic driver targeted vaccines with selective ICB offers a rational paradigm for precision immunotherapy, which we are clinically evaluating in a phase II trial (NCT03632941).

FGFR4 regulates tumor subtype differentiation in luminal breast cancer and metastatic disease.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.

B Cells and T Follicular Helper Cells Mediate Response to Checkpoint Inhibitors in High Mutation Burden Mouse Models of Breast Cancer.

This study identifies mechanisms mediating responses to immune checkpoint inhibitors using mouse models of triple-negative breast cancer. By creating new mammary tumor models, we find that tumor mutation burden and specific immune cells are associated with response. Further, we developed a rich resource of single-cell RNA-seq and bulk mRNA-seq data of immunotherapy-treated and non-treated tumors from sensitive and resistant murine models. Using this, we uncover that immune checkpoint therapy induces T follicular helper cell activation of B cells to facilitate the anti-tumor response in these models. We also show that B cell activation of T cells and the generation of antibody are key to immunotherapy response and propose a new biomarker for immune checkpoint therapy. In total, this work presents resources of new preclinical models of breast cancer with large mRNA-seq and single-cell RNA-seq datasets annotated for sensitivity to therapy and uncovers new components of response to immune checkpoint inhibitors.

E2F1 Drives Breast Cancer Metastasis by Regulating the Target Gene FGF13 and Altering Cell Migration.

In prior work we demonstrated that loss of E2F transcription factors inhibits metastasis. Here we address the mechanisms for this phenotype and identify the E2F regulated genes that coordinate tumor cell metastasis. Transcriptomic profiling of E2F1 knockout tumors identified a role for E2F1 as a master regulator of a suite of pro-metastatic genes, but also uncovered E2F1 target genes with an unknown role in pulmonary metastasis. High expression of one of these genes, Fgf13, is associated with early human breast cancer metastasis in a clinical dataset. Together these data led to the hypothesis that Fgf13 is critical for breast cancer metastasis, and that upregulation of Fgf13 may partially explain how E2F1 promotes breast cancer metastasis. To test this hypothesis we ablated Fgf13 via CRISPR. Deletion of Fgf13 in a MMTV-PyMT breast cancer cell line reduces colonization of the lungs in a tail vein injection. In addition, loss of Fgf13 reduced in vitro cell migration, suggesting that Fgf13 may be critical for tumor cells to escape the primary tumor and to colonize the distal sites. The significance of this work is twofold: we have both uncovered genomic features by which E2F1 regulates metastasis and we have identified new pro-metastatic functions for the E2F1 target gene Fgf13.

A mouse model featuring tissue-specific deletion of p53 and Brca1 gives rise to mammary tumors with genomic and transcriptomic similarities to human basal-like breast cancer.

PURPOSE AND METHODS: In human basal-like breast cancer, mutations and deletions in TP53 and BRCA1 are frequent oncogenic events. Thus, we interbred mice expressing the CRE-recombinase with mice harboring loxP sites at TP53 and BRCA1 (K14-Cre; p53f/f Brca1f/f) to test the hypothesis that tissue-specific deletion of TP53 and BRCA1 would give rise to tumors reflective of human basal-like breast cancer. RESULTS: In support of our hypothesis, these transgenic mice developed tumors that express basal-like cytokeratins and demonstrated intrinsic gene expression features similar to human basal-like tumors. Array comparative genomic hybridization revealed a striking conservation of copy number alterations between the K14-Cre; p53f/f Brca1f/f mouse model and human basal-like breast cancer. Conserved events included MYC amplification, KRAS amplification, and RB1 loss. Microarray analysis demonstrated that these DNA copy number events also led to corresponding changes in signatures of pathway activation including high proliferation due to RB1 loss. K14-Cre; p53f/f Brca1f/f also matched human basal-like breast cancer for a propensity to have immune cell infiltrates. Given the long latency of K14-Cre; p53f/f Brca1f/f tumors (~ 250 days), we created tumor syngeneic transplant lines, as well as in vitro cell lines, which were tested for sensitivity to carboplatin and paclitaxel. These therapies invoked acute regression, extended overall survival, and resulted in gene expression signatures of an anti-tumor immune response. CONCLUSION: These findings demonstrate that this model is a valuable preclinical resource for the study of human basal-like breast cancer.

Identification of Jun loss promotes resistance to histone deacetylase inhibitor entinostat through Myc signaling in luminal breast cancer.

BACKGROUND: Based on promising phase II data, the histone deacetylase inhibitor entinostat is in phase III trials for patients with metastatic estrogen receptor-positive breast cancer. Predictors of sensitivity and resistance, however, remain unknown. METHODS: A total of eight cell lines and nine mouse models of breast cancer were treated with entinostat. Luminal cell lines were treated with or without entinostat at their IC50 doses, and MMTV/Neu luminal mouse tumors were untreated or treated with entinostat until progression. We investigated these models using their gene expression profiling by microarray and copy number by arrayCGH. We also utilized the network-based DawnRank algorithm that integrates DNA and RNA data to identify driver genes of resistance. The impact of candidate drivers was investigated in The Cancer Genome Atlas and METABRIC breast cancer datasets. RESULTS: Luminal models displayed enhanced sensitivity to entinostat as compared to basal-like or claudin-low models. Both in vitro and in vivo luminal models showed significant downregulation of Myc gene signatures following entinostat treatment. Myc gene signatures became upregulated on tumor progression in vivo and overexpression of Myc conferred resistance to entinostat in vitro. Further examination of resistance mechanisms in MMTV/Neu tumors identified a portion of mouse chromosome 4 that had DNA copy number loss and low gene expression. Within this region, Jun was computationally identified to be a driver gene of resistance. Jun knockdown in cell lines resulted in upregulation of Myc signatures and made these lines more resistant to entinostat. Jun-deleted samples, found in 17-23% of luminal patients, had significantly higher Myc signature scores that predicted worse survival. CONCLUSIONS: Entinostat inhibited luminal breast cancer through Myc signaling, which was upregulated by Jun DNA loss to promote resistance to entinostat in our models. Jun DNA copy number loss, and/or high MYC signatures, might represent biomarkers for entinostat responsiveness in luminal breast cancer.

Cdh1 and Pik3ca Mutations Cooperate to Induce Immune-Related Invasive Lobular Carcinoma of the Breast.

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.

A Review of FOXI3 Regulation of Development and Possible Roles in Cancer Progression and Metastasis.

Development and cancer share a variety of functional traits such as EMT, cell migration, angiogenesis, and tissue remodeling. In addition, many cellular signaling pathways are noted to coordinate developmental processes and facilitate aspects of tumor progression. The Forkhead box superfamily of transcription factors consists of a highly conserved DNA binding domain, which binds to specific DNA sequences and play significant roles during adult tissue homoeostasis and embryogenesis including development, differentiation, metabolism, proliferation, apoptosis, migration, and invasion. Interestingly, various studies have implicated the role of key Fox family members such as FOXP, FOXO, and FOXA during cancer initiation and metastases. FOXI3, a member of the Forkhead family affects embryogenesis, development, and bone remodeling; however, no studies have reported a role in cancer. In this review, we summarize the role of FOXI3 in embryogenesis and bone development and discuss its potential involvement in cancer progression with a focus on the bone metastasis. Moreover, we hypothesize possible mechanisms underlying the role of FOXI3 in the development of solid tumor bone metastasis.

Integrated Molecular Characterization of Testicular Germ Cell Tumors.

We studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance-KIT, KRAS, and NRAS-exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas.

Histological subtypes of mouse mammary tumors reveal conserved relationships to human cancers.

Human breast cancer has been characterized by extensive transcriptional heterogeneity, with dominant patterns reflected in the intrinsic subtypes. Mouse models of breast cancer also have heterogeneous transcriptomes and we noted that specific histological subtypes were associated with particular subsets. We hypothesized that unique sets of genes define each tumor histological type across mouse models of breast cancer. Using mouse models that contained both gene expression data and expert pathologist classification of tumor histology on a sample by sample basis, we predicted and validated gene expression signatures for Papillary, EMT, Microacinar and other histological subtypes. These signatures predict known histological events across murine breast cancer models and identify counterparts of mouse mammary tumor types in subtypes of human breast cancer. Importantly, the EMT, Adenomyoepithelial, and Solid signatures were predictive of clinical events in human breast cancer. In addition, a pan-cancer comparison revealed that the histological signatures were active in a variety of human cancers such as lung, oral, and esophageal squamous tumors. Finally, the differentiation status and transcriptional activity implicit within these signatures was identified. These data reveal that within tumor histology groups are unique gene expression profiles of differentiation and pathway activity that stretch well beyond the transgenic initiating events and that have clear applicability to human cancers. As a result, our work provides a predictive resource and insights into possible mechanisms that govern tumor heterogeneity.

Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation.

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

A genomic analysis of mouse models of breast cancer reveals molecular features of mouse models and relationships to human breast cancer.

INTRODUCTION: Genomic variability limits the efficacy of breast cancer therapy. To simplify the study of the molecular complexity of breast cancer, researchers have used mouse mammary tumor models. However, the degree to which mouse models model human breast cancer and are reflective of the human heterogeneity has yet to be demonstrated with gene expression studies on a large scale. METHODS: To this end, we have built a database consisting of 1,172 mouse mammary tumor samples from 26 different major oncogenic mouse mammary tumor models. RESULTS: In this dataset we identified heterogeneity within mouse models and noted a surprising amount of interrelatedness between models, despite differences in the tumor initiating oncogene. Making comparisons between models, we identified differentially expressed genes with alteration correlating with initiating events in each model. Using annotation tools, we identified transcription factors with a high likelihood of activity within these models. Gene signatures predicted activation of major cell signaling pathways in each model, predictions that correlated with previous genetic studies. Finally, we noted relationships between mouse models and human breast cancer at both the level of gene expression and predicted signal pathway activity. Importantly, we identified individual mouse models that recapitulate human breast cancer heterogeneity at the level of gene expression. CONCLUSIONS: This work underscores the importance of fully characterizing mouse tumor biology at molecular, histological and genomic levels before a valid comparison to human breast cancer may be drawn and provides an important bioinformatic resource.

The E2F transcription factors regulate tumor development and metastasis in a mouse model of metastatic breast cancer.

While the E2F transcription factors (E2Fs) have a clearly defined role in cell cycle control, recent work has uncovered new functions. Using genomic signature methods, we predicted a role for the activator E2F transcription factors in the mouse mammary tumor virus (MMTV)-polyomavirus middle T oncoprotein (PyMT) mouse model of metastatic breast cancer. To genetically test the hypothesis that the E2Fs function to regulate tumor development and metastasis, we interbred MMTV-PyMT mice with E2F1, E2F2, or E2F3 knockout mice. With the ablation of individual E2Fs, we noted alterations of tumor latency, histology, and vasculature. Interestingly, we noted striking reductions in metastatic capacity and in the number of circulating tumor cells in both the E2F1 and E2F2 knockout backgrounds. Investigating E2F target genes that mediate metastasis, we found that E2F loss led to decreased levels of vascular endothelial growth factor (Vegfa), Bmp4, Cyr61, Nupr1, Plod 2, P4ha1, Adamts1, Lgals3, and Angpt2. These gene expression changes indicate that the E2Fs control the expression of genes critical to angiogenesis, the remodeling of the extracellular matrix, tumor cell survival, and tumor cell interactions with vascular endothelial cells that facilitate metastasis to the lungs. Taken together, these results reveal that the E2F transcription factors play key roles in mediating tumor development and metastasis in addition to their well-characterized roles in cell cycle control.