Guang Gao

Gain of Glucose-Independent Growth upon Metastasis of Breast Cancer Cells to the Brain

Breast cancer brain metastasis is resistant to therapy and a particularly poor prognostic feature in patient survival. Altered metabolism is a common feature of cancer cells, but little is known as to what metabolic changes benefit breast cancer brain metastases. We found that brain metastatic breast cancer cells evolved the ability to survive and proliferate independent of glucose due to enhanced gluconeogenesis and oxidations of glutamine and branched chain amino acids, which together sustain the nonoxidative pentose pathway for purine synthesis. Silencing expression of fructose-1,6-bisphosphatases (FBP) in brain metastatic cells reduced their viability and improved the survival of metastasis-bearing immunocompetent hosts. Clinically, we showed that brain metastases from human breast cancer patients expressed higher levels of FBP and glycogen than the corresponding primary tumors. Together, our findings identify a critical metabolic condition required to sustain brain metastasis and suggest that targeting gluconeogenesis may help eradicate this deadly feature in advanced breast cancer patients.

ATP Citrate Lyase Mediates Resistance of Colorectal Cancer Cells to SN38

Combination chemotherapy is standard for metastatic colorectal cancer; however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic colorectal cancer, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in colorectal cancer compared with its levels in normal mucosa and in chemoresistant colorectal cancer cells compared with isogenic chemo-naïve colorectal cancer cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve colorectal cancer cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve colorectal cancer cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in resensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully resensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance. Mol Cancer Ther; 12(12); 2782–91. ©2013 AACR.

Involvement of de novo synthesized palmitate and mitochondrial EGFR in EGF induced mitochondrial fusion of cancer cells

Increased expressions of fatty acid synthase (FASN) and epidermal growth factor receptor (EGFR) are common in cancer cells. De novo synthesis of palmitate by FASN is critical for the survival of cancer cells via mechanisms independent of its role as an energy substrate. Besides the plasma membrane and the nucleus, EGFR can also localize at the mitochondria; however, signals that can activate mitochondrial EGFR (mtEGFR) and the functions of mtEGFR of cancer cells remain unknown. The present study characterizes mtEGFR in the mitochondria of cancer cells (prostate and breast) and reveals that mtEGFR can promote mitochondrial fusion through increasing the protein levels of fusion proteins PHB2 and OPA1. Activation of plasma membranous EGFR (pmEGFR) stimulates the de novo synthesis of palmitate through activation of FASN and ATP-citrate lyase (ACLy). In vitro kinase assay with isolated mitochondria shows that palmitate can activate mtEGFR. Inhibition of FASN blocks the mtEGFR phosphorylation and palmitoylation induced by EGF. Mutational studies show that the cysteine 797 is important for mtEGFR activation and palmitoylation. Inhibition of FASN can block EGF induced mitochondrial fusion and increased the sensitivity of prostate cancer cells to EGFR tyrosine kinase inhibitor. In conclusion, these results suggest that mtEGFR can be activated by pmEGFR through de novo synthesized palmitate to promote mitochondrial fusion and survival of cancer cells. This mechanism may serve as a novel target to improve EGFR-based cancer therapy.

EGFR–SGLT1 interaction does not respond to EGFR modulators, but inhibition of SGLT1 sensitizes prostate cancer cells to EGFR tyrosine kinase inhibitors

Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis in malignant tumors. Sodium/glucose co‐transporter 1 (SGLT1) is an active glucose transporter that is overexpressed in many cancers including prostate cancer. Previously, we found that EGFR interacts with and stabilizes SGLT1 in cancer cells.

Sodium/Glucose Co-transporter 1 Expression Increases in Human Diseased Prostate

Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that takes up glucose into cells independent of the extracellular concentration of glucose. This transporter plays a critical role in maintaining glucose homeostasis at both physiological and pathological levels. The expression level of SGLT1 in normal and diseased human prostatic tissue has not been determined. We produced two rabbit polyclonal antibodies against human SGLT1, one each for immunohistochemical and Western blot analyses, and characterized the expression of SGLT1 in human prostate tissues: normal prostate (n=3), benign prostatic hyperplasia (BPH) (n=53), prostatic intraepithelial neoplasia (PIN) (n=9), and prostate cancer (PCa) (n=44). In normal prostate tissue, SGLT1 was weakly expressed exclusively in the epithelium. The transporter was significantly increased in the basal cells and stromal cells of BPH, increased in the epithelial cells of PIN, and frequently overexpressed in stromal cells and universally overexpressed in the tumor cells of PCa. The pattern of expression was shown as membranous/ cytoplasmic staining in low-grade cancer cells and nuclear envelope staining in high-grade cancer cells. The SGLT1-positive stromal cells of BPH and PCa tissues were negative for tenascin, a marker of reactive stromal cells. We concluded that SGLT1 is up-regulated in BPH and PCa, and SGLT1 may serve as a potential therapeutic target for treating these prostate disorders.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.A new inhibitor targeting the mitochondrial complex I shows antitumor activity in preclinical models of acute myeloid leukemia and glioblastoma relying on oxidative phosphorylation.

Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer

Lung cancer is a devastating disease that remains a top cause of cancer mortality. Despite improvements with targeted and immunotherapies, the majority of patients with lung cancer lack effective therapies, underscoring the need for additional treatment approaches. Genomic studies have identified frequent alterations in components of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A. To understand the mechanisms of tumorigenesis driven by mutations in this complex, we developed a genetically engineered mouse model of lung adenocarcinoma by ablating Smarca4 in the lung epithelium. We demonstrate that Smarca4 acts as a bona fide tumor suppressor and cooperates with p53 loss and Kras activation. Gene expression analyses revealed the signature of enhanced oxidative phosphorylation (OXPHOS) in SMARCA4 mutant tumors. We further show that SMARCA4 mutant cells have enhanced oxygen consumption and increased respiratory capacity. Importantly, SMARCA4 mutant lung cancer cell lines and xenograft tumors have marked sensitivity to inhibition of OXPHOS by a novel small molecule, IACS-010759, that is under clinical development. Mechanistically, we show that SMARCA4-deficient cells have a blunted transcriptional response to energy stress creating a therapeutically exploitable synthetic lethal interaction. These findings provide the mechanistic basis for further development of OXPHOS inhibitors as therapeutics against SWI/SNF mutant tumors.SMARCA4 loss in non-small-cell lung cancer creates a metabolic dependency on oxidative phosphorylation that can be targeted using a new small-molecule inhibitor.

Author Correction: Mutations in the SWI/SNF complex induce a targetable dependence on oxidative phosphorylation in lung cancer

In the version of this article originally published, information regarding several funding sources was omitted from the Acknowledgements section. The following sentences should have been included: “This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program, the UT Lung SPORE 5 P50 CA07090, and the MD Anderson Cancer Center Support Grant P30CA01667. V.P is supported by R01CA155196-01A1 from the National Cancer Institute.” Also, reference 18 was incorrect. The original reference was: Kim, E. S. et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov. 1, 44–53 (2011). It should have been: Papadimitrakopoulou, V. et al. The BATTLE-2 study: a biomarker-integrated targeted therapy study in previously treated patients with advanced non–small-cell lung cancer. J Clin. Oncol. 34, 3638–3647 (2016). The errors have been corrected in the HTML and PDF versions of this article.

Abstract 3016: Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models

Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing disease associated with less than 10% 5-year survival rate. Various treatment regimens failed to improve survival of PDAC patients, thus a critical need exists to identify druggable targets essential for tumor maintenance. We developed a powerful in vivo platform that enables the identification of new molecular drivers in the PDAC context where activating mutation of KRAS gene and loss of p53 dominate the genetic landscape. Through an in vivo loss of function screen performed in KRAS/p53 mutant PDAC primary patient models, we identify protein arginine methyltransferase 1 (PRMT1) as top scoring hit. This novel dependency in PDAC was subsequently validated in multiple PDAC models using both shRNA mediated as well as CRISPR base genetic inhibition and we demonstrated that PRMT1 knockdown induces a significant growth inhibition in vitro. Methylation of arginine 3 on histone H4 (H4R3me2a) as well as global arginine methylation was also evaluated and showed a dramatic reduction upon PRMT1 knockdown, correlating observed phenotype with target engagement. To further confirm a role for PRMT1 in tumor maintenance, we developed inducible PRMT1 knockdown in a primary patient model and showed a dramatic tumor growth inhibition (TGI) in vivo upon PRMT1 knockdown. PRMT1 is the primary enzyme responsible for arginine asymmetric demethylation, however other members of the Type I family are also involved in this process and we evaluated the role of protein arginine methyltransferase 4 (PRMT4) and 6 (PRMT6) in our workhorse model. Surprisingly, no significant phenotypic response was observed upon genetic inhibition of PRMT4 or PRMT6 suggesting no redundancy between different PRMT type I and a unique dependency on PRMT1. To strengthen and complement the genetic validation, we leveraged a PRMT Type I inhibitor and confirmed in vitro results as well as in vivo efficacy at tolerated doses (xenograft vs allograft). Key models have been prioritized in order to inform on PRMT1 dependency and to refine responder population. Our research has identified and validated for the first time an arginine methyltransferase as a novel genetic vulnerability in PDAC and strongly suggest PRMT1 as a new therapeutic opportunity in PDAC cancers. Citation Format: Virginia Giuliani, Bhavatarini Vangamudi, Erika Suzuki, Meredith Miller, Chiu-Yi Liu, Alessandro Carugo, Christopher Bristow, Guang Gao, Jing Han, Yuting Sun, Ningping Feng, Edward Chang, Joseph Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Carlo Toniatti, Timothy Heffernan, Philip Jones, Giulio Draetta. Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2017-3016

Abstract B64: Development of a novel strategy to overcome drug resistance by targeting ATP citrate lyase and de novo lipogenesis in colorectal cancer cells

Background: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Although the response rate to current systemic therapies is ∼50%, drug resistance develops in nearly all patients leading to 50,000 deaths each year. Overcoming drug resistance involves understanding the mechanisms by which cancer cells adapt to the genotoxic stress. Metabolic changes at levels of mitochondria function, glucose metabolism, and de novo synthesis of fatty acids frequently occurs in malignant cells and impacts tumor development and growth. Our laboratory established in vitro drug-resistant models of CRC cells by chronic exposure of HT29 cells to increasing doses of chemotherapeutic agents (oxaliplatin, 5-FU and SN38) over a period of 4–6 months. We recently reported a metabolic switch to the glycolytic phenotype due to mitochondria defects in the oxaliplatin-resistant CRC cells (HT29-OXR) (Zhou et al, Cancer Research, 2012). In this study, we tested the hypothesis that deregulation of de novo lipogenesis pathways plays an important role in chemoresistance of CRC cells. Methods: A previous study (Bose et al. Br J Ca, 2011) using unbiased proteomic profiling by mass spectroscopy (MS) was used to determine the proteomic signature of defective metabolic pathways in the oxaliplatin-resistant CRC cells (OXR cells). ATP-citrate-lyase (ACLy), the key enzyme of de novo lipogenesis pathway, was examined for protein levels and activation. The lipid content of resistant cells was examined by transmission electron microscope (TEM) and Oil Red staining. Transient knockdown of the ACLy protein by siRNA was used to study re-chemosensitization of the resistant cells. A small molecule inhibitor of ACLy was studied in combination with chemotherapeutic agents in resistant CRC cells for growth inhibitory (MTT assay) and cytotoxic effects (PARP cleavage and Annexin V staining). A cell free assay was used to test the potency of the ACLy inhibitor on ACLy activation. Results: Activated ACLy protein level (phosphorylation on S454) was demonstrated by Western blot analysis in the HT29-OXR and SN38-resistant (HT29-SNR) cells, but not in 5-FU-resistant (HT29-FUR) cells. The OxR cells showed a 2–3 fold increase in lipid droplets numbers (by TEM examination) and fatty acid content (by Oil Red staining) than the parental cells. Furthermore, transient knockdown of the ACLy protein by siRNA demonstrated a return to chemosensitization when cells were treated with oxaliplatin. IC50 values of the ACLy inhibitor for parental HT29 and HT29-OXR, -SNR and —FUR cells were ∼30μM. As a single agent, the ACLy inhibitor blocked phosphorylation of ACLy and induced apoptosis in a concentration-dependent manner in parental HT29 cells, and its resistant derivatives -OXR and -SNR cells. Combination of the ACLy inhibitor at concentrations sufficient to block ACLy phosphorylation with oxaliplatin and SN38 showed enhanced effects on growth inhibition (MTT) and apoptosis induction (PARP cleavage and Annexin V assay) in HT29 cells-OXR and -SNR cells. Conclusions: Chemoresistant CRC cells demonstrated: 1) increased de novo lipogenesis, 2) elevated levels of key lipogenesis enzymes ACLy, 3) dependence on ACLy activity for cell survival under cytotoxic stress. This metabolic switch likely contributes to the chemoresistant phenotype of CRC cells. Targeting an early step of de novo lipogenesis such as blocking ACLy activity may provide a novel strategy to overcome drug-resistance in CRC cells.