Casey Frankenberger

HMGA2/TET1/HOXA9 signaling pathway regulates breast cancer growth and metastasis

The ten–eleven translocation (TET) family of methylcytosine dioxygenases initiates demethylation of DNA and is associated with tumorigenesis in many cancers; however, the mechanism is mostly unknown. Here we identify upstream activators and downstream effectors of TET1 in breast cancer using human breast cancer cells and a genetically engineered mouse model. We show that depleting the architectural transcription factor high mobility group AT-hook 2 (HMGA2) induces TET1. TET1 binds and demethylates its own promoter and the promoter of homeobox A (HOXA) genes, enhancing its own expression and stimulating expression of HOXA genes including HOXA7 and HOXA9. Both TET1 and HOXA9 suppress breast tumor growth and metastasis in mouse xenografts. The genes comprising the HMGA2–TET1–HOXA9 pathway are coordinately regulated in breast cancer and together encompass a prognostic signature for patient survival. These results implicate the HMGA2–TET1–HOX signaling pathway in the epigenetic regulation of human breast cancer and highlight the importance of targeting methylation in specific subpopulations as a potential therapeutic strategy.

Signalling pathway for RKIP and Let‐7 regulates and predicts metastatic breast cancer

Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let‐7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let‐7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let‐7‐regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.

Mitophagy defects arising from BNip3 loss promote mammary tumor progression to metastasis

BNip3 is a hypoxia‐inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif‐1α and Hif target genes, including those involved in glycolysis and angiogenesis—two processes that are also markedly increased in BNip3‐null tumors. Glycolysis inhibition attenuates the growth of BNip3‐null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple‐negative breast cancer (TNBC). These studies show that mitochondrial dysfunction—caused by defects in mitophagy—can promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

Network of mutually repressive metastasis regulators can promote cell heterogeneity and metastatic transitions.

Significance Cancer progression, as an evolutionary process, should accelerate if higher cellular variability is present. However, the sources of nongenetic variability in metastatic progression are largely unknown. To address this question, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we reveal a network architecture between the metastasis suppressor RKIP and the metastasis promoter BACH1 that enables single cells to generate a stable subpopulation of prometastatic cells without any genetic changes. The sources and consequences of nongenetic variability in metastatic progression are largely unknown. To address these questions, we characterized a transcriptional regulatory network for the metastasis suppressor Raf kinase inhibitory protein (RKIP). We previously showed that the transcription factor BACH1 is negatively regulated by RKIP and promotes breast cancer metastasis. Here we demonstrate that BACH1 acts in a double-negative (overall positive) feedback loop to inhibit RKIP transcription in breast cancer cells. BACH1 also negatively regulates its own transcription. Analysis of the BACH1 network reveals the existence of an inverse relationship between BACH1 and RKIP involving both monostable and bistable transitions that can potentially give rise to nongenetic variability. Single-cell analysis confirmed monostable and bistable-like behavior. Treatment with histone deacetylase inhibitors or depletion of the polycomb repressor enhancer of zeste homolog 2 altered relative RKIP and BACH1 levels in a manner consistent with a prometastatic state. Together, our results suggest that the mutually repressive relationship between metastatic regulators such as RKIP and BACH1 can play a key role in determining metastatic progression in cancer.

RKIP and HMGA2 regulate breast tumor survival and metastasis through lysyl oxidase and syndecan-2

Elucidating targets of physiological tumor metastasis suppressors can highlight key signaling pathways leading to invasion and metastasis. To identify downstream targets of the metastasis suppressor Raf-1 kinase inhibitory protein (RKIP/PEBP1), we utilized an integrated approach based upon statistical analysis of tumor gene expression data combined with experimental validation. Previous studies from our laboratory identified the architectural transcription factor and oncogene, high mobility group AT-hook 2 (HMGA2), as a target of inhibition by RKIP. Here we identify two signaling pathways that promote HMGA2-driven metastasis. Using both human breast tumor cells and an MMTV-Wnt mouse breast tumor model, we show that RKIP induces and HMGA2 inhibits expression of miR-200b; miR-200b directly inhibits expression of lysyl oxidase (LOX), leading to decreased invasion. RKIP also inhibits syndecan-2 (SDC2), which is aberrantly expressed in breast cancer, via downregulation of HMGA2; but this mechanism is independent of miR-200. Depletion of SDC2 induces apoptosis and suppresses breast tumor growth and metastasis in mouse xenografts. RKIP, LOX and SDC2 are coordinately regulated and collectively encompass a prognostic signature for metastasis-free survival in ER-negative breast cancer patients. Taken together, our findings reveal two novel signaling pathways targeted by the metastasis suppressor RKIP that regulate remodeling of the extracellular matrix and tumor survival.

Metastasis Suppressors Regulate the Tumor Microenvironment by Blocking Recruitment of Prometastatic Tumor-Associated Macrophages

Triple-negative breast cancer (TNBC) patients have the highest risk of recurrence and metastasis. Because they cannot be treated with targeted therapies, and many do not respond to chemotherapy, they represent a clinically underserved group. TNBC is characterized by reduced expression of metastasis suppressors such as Raf kinase inhibitory protein (RKIP), which inhibits tumor invasiveness. Mechanisms by which metastasis suppressors alter tumor cells are well characterized; however, their ability to regulate the tumor microenvironment and the importance of such regulation to metastasis suppression are incompletely understood. Here, we use species-specific RNA sequencing to show that RKIP expression in tumors markedly reduces the number and metastatic potential of infiltrating tumor-associated macrophages (TAM). TAMs isolated from nonmetastatic RKIP(+) tumors, relative to metastatic RKIP(-) tumors, exhibit a reduced ability to drive tumor cell invasion and decreased secretion of prometastatic factors, including PRGN, and shed TNFR2. RKIP regulates TAM recruitment by blocking HMGA2, resulting in reduced expression of numerous macrophage chemotactic factors, including CCL5. CCL5 overexpression in RKIP(+) tumors restores recruitment of prometastatic TAMs and intravasation, whereas treatment with the CCL5 receptor antagonist Maraviroc reduces TAM infiltration. These results highlight the importance of RKIP as a regulator of TAM recruitment through chemokines such as CCL5. The clinical significance of these interactions is underscored by our demonstration that a signature comprised of RKIP signaling and prometastatic TAM factors strikingly separates TNBC patients based on survival outcome. Collectively, our findings identify TAMs as a previously unsuspected mechanism by which the metastasis-suppressor RKIP regulates tumor invasiveness, and further suggest that TNBC patients with decreased RKIP activity and increased TAM infiltration may respond to macrophage-based therapeutics.

A Prognostic Gene Signature for Metastasis-Free Survival of Triple Negative Breast Cancer Patients

Although triple negative breast cancers (TNBC) are the most aggressive subtype of breast cancer, they currently lack targeted therapies. Because this classification still includes a heterogeneous collection of tumors, new tools to classify TNBCs are urgently required in order to improve our prognostic capability for high risk patients and predict response to therapy. We previously defined a gene expression signature, RKIP Pathway Metastasis Signature (RPMS), based upon a metastasis-suppressive signaling pathway initiated by Raf Kinase Inhibitory Protein (RKIP). We have now generated a new BACH1 Pathway Metastasis gene signature (BPMS) that utilizes targets of the metastasis regulator BACH1. Specifically, we substituted experimentally validated target genes to generate a new BACH1 metagene, developed an approach to optimize patient tumor stratification, and reduced the number of signature genes to 30. The BPMS significantly and selectively stratified metastasis-free survival in basal-like and, in particular, TNBC patients. In addition, the BPMS further stratified patients identified as having a good or poor prognosis by other signatures including the Mammaprint® and Oncotype® clinical tests. The BPMS is thus complementary to existing signatures and is a prognostic tool for high risk ER-HER2- patients. We also demonstrate the potential clinical applicability of the BPMS as a single sample predictor. Together, these results reveal the potential of this pathway-based BPMS gene signature to identify high risk TNBC patients that can respond effectively to targeted therapy, and highlight BPMS genes as novel drug targets for therapeutic development.

Anti-tumor effects of an engineered "killer" transfer RNA.

A hallmark of cancer cells is their ability to continuously divide; and rapid proliferation requires increased protein translation. Elevating levels of misfolded proteins can elicit growth arrest due to ER stress and decreased global translation. Failure to correct prolonged ER stress eventually results in cell death via apoptosis. tRNA(Ser)(AAU) is an engineered human tRNA(Ser) with an anticodon coding for isoleucine. Here we test the possibility that tRNA(Ser)(AAU) can be an effective killing agent of breast cancer cells and can effectively inhibit tumor-formation in mice. We found that tRNA(Ser)(AAU) exert strong effects on breast cancer translation activity, cell viability, and tumor formation. Translation is strongly inhibited by tRNA(Ser)(AAU) in both tumorigenic and non-tumorigenic cells. tRNA(Ser)(AAU) significantly decreased the number of viable cells over time. A short time treatment with tRNA(Ser)(AAU) was sufficient to eliminate breast tumor formation in a xenograft mouse model. Our results indicate that tRNA(Ser)(AAU) can inhibit breast cancer metabolism, growth and tumor formation. This RNA has strong anti-cancer effects and presents an opportunity for its development into an anti-tumor agent. Because tRNA(Ser)(AAU) corrupts the protein synthesis mechanism that is an integral component of the cell, it would be extremely difficult for tumor cells to evolve and develop resistance against this anti-tumor agent.

Gene expression in local stroma reflects breast tumor states and predicts patient outcome

The surrounding microenvironment has been implicated in the progression of breast tumors to metastasis. However, the degree to which metastatic breast tumors locally reprogram stromal cells as they disrupt tissue boundaries is not well understood. We used species-specific RNA sequencing in a mouse xenograft model to determine how the metastasis suppressor RKIP influences transcription in a panel of paired tumor and stroma tissues. We find that gene expression in metastatic breast tumors is pervasively correlated with gene expression in local stroma of both mouse xenografts and human patients. Changes in stromal gene expression elicited by tumors better predicts subtype and patient survival than tumor gene expression, and genes with coordinated expression in both tissues predict metastasis-free survival. These observations support the use of stroma-based strategies for the diagnosis and prognosis of breast cancer.

Abstract B15: Metastatic breast tumors regulate gene expression at distal mammary sites that predicts patient outcome

The molecular interactions between cancer and stromal cells within the tumor microenvironment enable tumor invasion, intravasation, and metastasis at distant sites. However, the degree to which metastatic breast tumors reprogram stromal cells both locally and at distant mammary tissues is not well understood. To address this question, we used species-specific RNA sequencing in a mouse xenograft model to determine how the metastasis suppressor RKIP influences transcription in tumor and stroma tissues. Here we show that metastatic tumors prime mammary tissue at a distant site in a manner that reflects local stromal responses. In addition, gene expression in metastatic breast tumors is pervasively correlated with gene expression in local stroma of both mouse xenografts and human patients. Changes in local and distant stromal gene expression elicited by metastatic tumors are better predictors of subtype and patient survival than tumor gene expression, supporting the use of stromal-based strategies for the diagnosis and prognosis of breast cancer. One mechanism by which changes at contralateral distal mammary breast occur is through exosomes secreted by tumor cells. These results indicate that tumors prime contralateral mammary tissue in a manner that reflects local stromal changes and predicts metastatic disease. This study has future application to our understanding of contralateral breast cancer. Citation Format: Jiyoung Lee, Russell Bainer, Casey Frankenberger, Daniel Rabe, sadiq Saleh, Morag Park, Gary An, Yoav Gilad, Marsha Rich Rosner. Metastatic breast tumors regulate gene expression at distal mammary sites that predicts patient outcome. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B15.