Brittany Anderton

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor–, progesterone receptor– or human epidermal growth factor 2 receptor–positive (RP) breast cancer. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient–derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer.

MicroRNA-494 within an oncogenic microRNA megacluster regulates G1/S transition in liver tumorigenesis through suppression of mutated in colorectal cancer.

Hepatocellular carcinoma (HCC) is associated with poor survival for patients and few effective treatment options, raising the need for novel therapeutic strategies. MicroRNAs (miRNAs) play important roles in tumor development and show deregulated patterns of expression in HCC. Because of the livers unique affinity for small nucleic acids, miRNA‐based therapy has been proposed in the treatment of liver disease. Thus, there is an urgent need to identify and characterize aberrantly expressed miRNAs in HCC. In our study, we profiled miRNA expression changes in de novo liver tumors driven by MYC and/or RAS, two canonical oncogenes activated in a majority of human HCCs. We identified an up‐regulated miRNA megacluster comprised of 53 miRNAs on mouse chromosome 12qF1 (human homolog 14q32). This miRNA megacluster is up‐regulated in all three transgenic liver models and in a subset of human HCCs. An unbiased functional analysis of all miRNAs within this cluster was performed. We found that miR‐494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the Mutated in Colorectal Cancer tumor suppressor. miR‐494 inhibition in human HCC cell lines decreases cellular transformation, and anti‐miR‐494 treatment of primary MYC‐driven liver tumor formation significantly diminishes tumor size. Conclusion: Our findings identify a new therapeutic target (miR‐494) for the treatment of HCC. (Hepatology 2014;58:202–215)

Cullin 1 Functions as a Centrosomal Suppressor of Centriole Multiplication by Regulating Polo-like Kinase 4 Protein Levels

Abnormal centrosome and centriole numbers are frequently detected in tumor cells where they can contribute to mitotic aberrations that cause chromosome missegregation and aneuploidy. The molecular mechanisms of centriole overduplication in malignant cells, however, are poorly characterized. Here, we show that the core SKP1-cullin-F-box component cullin 1 (CUL1) localizes to maternal centrioles and that CUL1 is critical for suppressing centriole overduplication through multiplication, a recently discovered mechanism whereby multiple daughter centrioles form concurrently at single maternal centrioles. We found that this activity of CUL1 involves the degradation of Polo-like kinase 4 (PLK4) at maternal centrioles. PLK4 is required for centriole duplication and strongly stimulates centriole multiplication when aberrantly expressed. We found that CUL1 is critical for the degradation of active PLK4 following deregulation of cyclin E/cyclin-dependent kinase 2 activity, as is frequently observed in human cancer cells, as well as for baseline PLK4 protein stability. Collectively, our results suggest that CUL1 may function as a tumor suppressor by regulating PLK4 protein levels and thereby restraining excessive daughter centriole formation at maternal centrioles.

PIM1 kinase inhibition as a targeted therapy against triple-negative breast tumors with elevated MYC expression

Triple-negative breast cancer (TNBC), in which cells lack expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, is the breast cancer subtype with the poorest outcome. No targeted therapy is available against this subtype of cancer owing to a lack of validated molecular targets. We previously reported that signaling involving MYC—an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes—is disproportionally higher in triple-negative (TN) tumors than in receptor-positive (RP) tumors. Direct inhibition of the oncogenic transcriptional activity of MYC has been challenging to achieve. Here, by conducting a shRNA screen targeting the kinome, we identified PIM1, a non-essential serine–threonine kinase, in a synthetic lethal interaction with MYC. PIM1 expression was higher in TN tumors than in RP tumors and was associated with poor prognosis in patients with hormone- and HER2-negative tumors. Small-molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic mouse models of breast cancer by inhibiting the oncogenic transcriptional activity of MYC and restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that have elevated MYC expression.

Taking on challenging targets: making MYC druggable.

The transcription factor proto-oncogene c-MYC (hereafter MYC) was first identified more than 3 decades ago and has since been found deregulated in a wide variety of the most aggressive human malignancies. As a pleiotropic transcription factor, MYC directly or indirectly controls expression of hundreds of coding and noncoding genes, which affect cell cycle entry, proliferation, differentiation, metabolism, and death/survival decisions of normal and cancer cells. Tumors with elevated MYC expression often exhibit highly proliferative, aggressive phenotypes, and elevated MYC expression has been correlated with diminished disease-free survival for a variety of human cancers. The use of MYC overexpression or MYC-dependent transcriptional gene signatures as clinical biomarkers is currently being investigated. Furthermore, preclinical animal and cell-based model systems have been extensively utilized in an effort to uncover the mechanisms of MYC-dependent tumorigenesis and tumor maintenance. Despite our ever-growing understanding of MYC biology, currently no targeted therapeutic strategy is clinically available to treat tumors that have acquired elevated MYC expression. This article summarizes the progresses being made to discover and implement new therapies to kill MYC over-expressing tumors-a target that was once deemed undruggable.

MYC-driven inhibition of the glutamate-cysteine ligase promotes glutathione depletion in liver cancer

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC‐driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl‐tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC‐driven tumors. We find that fewer glutamine‐derived carbons are incorporated into GSH in tumor tissue relative to non‐tumor tissue. Expression of GCLC, the rate‐limiting enzyme of GSH synthesis, is attenuated by the MYC‐induced microRNA miR‐18a. Inhibition of miR‐18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC‐driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC‐dependent attenuation of GCLC by miR‐18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.

Hybrid thematic analysis reveals themes for assessing student understanding of biotechnology

Abstract Despite efforts to increase teaching of biotechnology worldwide, there are concerns that public literacy of genetic technologies remains insufficient. Improved education strategies are expected to empower individuals to make informed decisions about biotechnology. To evaluate the teaching and learning of this complex topic, qualitative assessment tools are needed. In this case study, we performed a hybrid thematic analysis to identify a set of overarching themes that can be used to evaluate individuals’ understanding of genetic technologies. We analysed the written justifications students gave for their attitudes on a range of genetic technologies, before and after peer-led discussion of each topic. We identified seven themes commonly detected in student responses, five of which have been previously described in studies of mass media communication of biotechnology. Our preliminary analysis suggests that peer-led discourse can promote changes in student understanding of biotechnology. We conclude that hybrid thematic analysis is a useful approach for evaluating the teaching and learning of genetic technologies. We discuss the utility of the hybrid approach and the themes described here for future studies of biotechnology education.

Semantic Network Analysis Reveals Opposing Online Representations of the Search Term “GMO”

Abstract Making sound food and agriculture decisions is important for global society and the environment. Experts tend to view crop genetic engineering, a technology that can improve yields and minimize impacts on the environment, more favorably than the public. Because there is a causal relationship between public opinion and public policy, it is important to understand how opinions about genetically engineered (GE) crops are influenced. The public increasingly seeks science information on the Internet. Here, semantic network analysis is performed to characterize the presentation of the term “GMO (genetically modified organism),” a proxy for food developed from GE crops, on the web. Texts from three sources are analyzed: U.S. federal websites, top pages from a Google search, and online news titles. We found that the framing and sentiment (positive, neutral, or negative attitudes) of “GMO” varies across these sources. It is described how differences in the portrayal of GE food by each source might affect public opinion. A current understanding of the types of information individuals may encounter online can provide insight into public opinion toward GE food. In turn, this knowledge can guide teaching and communication efforts by the scientific community to promote informed decision‐making about agricultural biotechnologies.

Abstract B34: PIM kinase as a novel therapeutic target for triple-negative breast cancer

The greatest clinical challenge in treating breast cancer occurs in those patients whose tumors lack expression of the estrogen and progesterone receptors and that of the HER2 oncoprotein. No targeted therapeutic strategies currently exist against this aggressive type of “triple negative” breast cancer (TNBC) due to lack of validated targets. We previously found that MYC mRNA, protein, and its signaling were disproportionally elevated in TN compared to receptor positive (RP) breast cancer. We sought to take advantage of the unique molecular feature found in this tumor type to identify potent and effective treatment strategies. Since MYC is an oncogenic transcription factor, rationally designed small molecule inhibitors that can directly inhibit its activity are not available for clinical use. An alternative approach to selectively kill MYC-driven tumors is to inhibit those proteins that are indispensable for the viability of such tumors, but are not essential in non-tumorigenic cells. This form of “indirect” treatment strategy has become known as the “synthetic-lethal” approach. To identify novel targets that are readily druggable for treating MYC-driven TNBC, we conducted a kinome MYC synthetic lethal shRNA screen in non-immortalized human mammary epithelial cells expressing a 4-hydroxytamoxifen (TAM)-activatable MycER transgene (HMEC-MycER). Of 600 human kinases targeted by 2,000 individual shRNA clones, 9 kinases were identified as hits as they were essential specifically for the MYC-activated HMEC cells. Among these hits, we focused on PIM1, a non-essential kinase, the knock-down of which had the greatest efficacy in causing cell death in the MYC-activated cells and had minimum inhibitory effect on the growth of the control cells. We determined that PIM1 expression was elevated in TN tumors and was associated with poor prognosis specifically in patients with hormone receptor-negative tumors. Small molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in a patient-derived tumor xenograft (PDX) mouse model by inhibiting oncogenic transcriptional activity of MYC while simultaneously restoring the function of the endogenous cell cycle inhibitor p27. Thus, our findings warrant clinical evaluation of small molecule PIM kinase inhibitors in patients with TN tumors that exhibit elevated MYC expression. Note: This abstract was not presented at the conference. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Sanjeev Balakrishnan, Kai Kessenbrock, Devon A. Lawson, Roman Camarda, Brittany N. Anderton, Alexey V. Bazarov, Henok Eyob, Julia Rohrberg, Paul Yaswen, Michael T. McManus, Hope S. Rugo, Zena Werb, Andrei Goga. PIM kinase as a novel therapeutic target for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B34.

Abstract 2673: Inhibition of fatty-acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC) compared to estrogen, progesterone and/or human epidermal growth factor 2 receptor-positive (RP) breast tumors. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains largely unexplored. We hypothesized that pharmacologic inhibition of MYC-driven metabolic pathways may serve as a therapeutic strategy for this clinically challenging subtype of breast cancer. Using a targeted metabolomics approach, we identified fatty-acid oxidation (FAO) intermediates as dramatically upregulated in a MYC-driven model of TNBC. A lipid metabolism gene signature was identified in patients with TNBC in the TCGA and multiple other clinical datasets, implicating FAO as a dysregulated pathway critical for TNBC metabolism. We find that MYC-overexpressing TNBC, including a transgenic model and patient-derived xenograft (PDX), display increased bioenergetic reliance upon FAO. Pharmacologic inhibition of FAO catastrophically decreases energy metabolism of MYC-overexpressing breast cancer, blocks growth of a MYC-driven transgenic TNBC model and MYC-overexpressing PDX. Our results demonstrate that inhibition of FAO is a novel therapeutic strategy against TNBCs that overexpress MYC. Citation Format: Roman Camarda, Alicia Y. Zhou, Rebecca A. Kohnz, Sanjeev Balakrishnan, Celine Mahieu, Brittany Anderton, Henok Eyob, Shingo Kajimura, Aaron Tward, Gregor Krings, Daniel K. Nomura, Andrei Goga. Inhibition of fatty-acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2673.