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.

Chemotherapy Coupled to Macrophage Inhibition Induces T-cell and B-cell Infiltration and Durable Regression in Triple-Negative Breast Cancer.

Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful antitumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small-molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single-cell RNA sequencing characterized tumor-infiltrating lymphocytes including Th cells and antigen-presenting B cells that were highly enriched in responders to combination therapy. In one model that exhibited long-term posttreatment tumor regression, high-dimensional imaging techniques identified the close spatial localization of B220+/CD86+-activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks posttreatment. The transcriptional and metabolic heterogeneity of TAMs was also characterized in two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model was highly conserved in human claudin-low breast cancers, and high expression of the TAM signature correlated with reduced overall survival in patients with breast cancer. This TAM signature may help identify human patients with claudin-low breast cancer that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rational immunotherapy combinations. SIGNIFICANCE: Immunostimulatory chemotherapy combined with pharmacologic inhibition of TAMs results in durable treatment responses elicited by Th cells and B cells in claudin-low TNBC models.

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.

Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFßR2 signaling pathway.

BRCA1 deficient breast cancers are aggressive and chemoresistant due, in part, to their enrichment of cancer stem cells that can be generated from carcinoma cells by an epithelial-mesenchymal transition (EMT). We previously discovered that BRCA1 deficiency activates EMT in mammary tumorigenesis. How BRCA1 controls EMT and how to effectively target BRCA1-deficient cancers remain elusive. We analyzed murine and human tumors and identified a role for Tgfßr2 in governing the molecular aspects of EMT that occur with Brca1 loss. We utilized CRISPR to delete Tgfßr2 and specific inhibitors to block Tgfßr2 activity and followed up with the molecular analysis of assays for tumor growth and metastasis. We discovered that heterozygous germline deletion, or epithelia-specific deletion of Brca1 in mice, activates Tgfßr2 signaling pathways in mammary tumors. BRCA1 depletion promotes TGFß-mediated EMT activation in cancer cells. BRCA1 binds to the TGFßR2 locus to repress its transcription. Targeted deletion or pharmaceutical inhibition of Tgfßr2 in Brca1-deficient tumor cells reduces EMT and suppresses tumorigenesis and metastasis. BRCA1 and TGFßR2 expression levels are inversely related in human breast cancers. This study reveals for the first time that a targetable TGFßR signaling pathway is directly activated by BRCA1-deficiency in the induction of EMT in breast cancer progression.

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.

B cells secrete GABA, which provokes a pro-tumor immune microenvironment.

In a recent publication in Nature, Zhang et al. report that foreign antigen stimulation elicits bountiful changes in lymphatic metabolite production-changes that include B cells secreting GABA, which reprograms macrophages and limits T cell cytotoxicity. This signifies a new mechanism by which B cells regulate immune suppression and facilitate tumor progression.

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.

NCOA5 deficiency promotes a unique liver protumorigenic microenvironment through p21WAF1/CIP1 overexpression, which is reversed by metformin.

Prevention and treatment options for hepatocellular carcinoma (HCC) are presently limited, underscoring the necessity for further elucidating molecular mechanisms underlying HCC development and identifying new prevention and therapeutic targets. Here, we demonstrate a unique protumorigenic niche in the livers of Ncoa5+/- mouse model of HCC, which is characterized by altered expression of a subset of genes including p21WAF1/CIP1 and proinflammatory cytokine genes, increased putative hepatic progenitors, and expansions of activated and tissue-resident memory (TRM) CD8+ T lymphocytes, myeloid-derived suppressor cells (MDSCs), and alternatively activated M2 macrophages. Importantly, prophylactic metformin treatment reversed these characteristics including aberrant p21WAF1/CIP1 expression and subsequently reduced HCC incidence in Ncoa5+/- male mice. Heterozygous deletion of the p21WAF1/CIP1 gene alleviated the key features associated with the protumorigenic niche in the livers of Ncoa5+/- male mice. Moreover, transcriptomic analysis reveals that preneoplastic livers of Ncoa5+/- mice are similar to the livers of nonalcoholic steatohepatitis patients as well as the adjacent noncancerous liver tissues of a subset of HCC patients with a relatively poor prognosis. Together, our results suggest that p21WAF1/CIP1 overexpression is essential in the development of protumorigenic microenvironment induced by NCOA5 deficiency and metformin prevents HCC development via alleviating p21WAF1/CIP1 overexpression and protumorigenic microenvironment.

Virus expression detection reveals RNA-sequencing contamination in TCGA.

BACKGROUND: Contamination of reagents and cross contamination across samples is a long-recognized issue in molecular biology laboratories. While often innocuous, contamination can lead to inaccurate results. Cantalupo et al., for example, found HeLa-derived human papillomavirus 18 (H-HPV18) in several of The Cancer Genome Atlas (TCGA) RNA-sequencing samples. This work motivated us to assess a greater number of samples and determine the origin of possible contaminations using viral sequences. To detect viruses with high specificity, we developed the publicly available workflow, VirDetect, that detects virus and laboratory vector sequences in RNA-seq samples. We applied VirDetect to 9143 RNA-seq samples sequenced at one TCGA sequencing center (28/33 cancer types) over 5 years. RESULTS: We confirmed that H-HPV18 was present in many samples and determined that viral transcripts from H-HPV18 significantly co-occurred with those from xenotropic mouse leukemia virus-related virus (XMRV). Using laboratory metadata and viral transcription, we determined that the likely contaminant was a pool of cell lines known as the "common reference", which was sequenced alongside TCGA RNA-seq samples as a control to monitor quality across technology transitions (i.e. microarray to GAII to HiSeq), and to link RNA-seq to previous generation microarrays that standardly used the "common reference". One of the cell lines in the pool was a laboratory isolate of MCF-7, which we discovered was infected with XMRV; another constituent of the pool was likely HeLa cells. CONCLUSIONS: Altogether, this indicates a multi-step contamination process. First, MCF-7 was infected with an XMRV. Second, this infected cell line was added to a pool of cell lines, which contained HeLa. Finally, RNA from this pool of cell lines contaminated several TCGA tumor samples most-likely during library construction. Thus, these human tumors with H-HPV or XMRV reads were likely not infected with H-HPV 18 or XMRV.

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.