Xiaohe Liu

5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors

Background: 5′-AMP-activated protein kinase (AMPK) is a ubiquitous regulator of cellular energy homeostasis. Results: AMPK contributes to the growth and core glucose metabolism of aggressive (rapidly growing) experimental human tumors. Conclusion: AMPK can be activated by metabolic stress to support the growth of aggressive tumors. Significance: AMPKs role in tumor biology could be strongly influenced by microenvironmental stress. Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [13C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.

A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Therapy

ABSTRACT A major goal in HIV eradication research is characterizing the reservoir cells that harbor HIV in the presence of antiretroviral therapy (ART), which reseed viremia after treatment is stopped. In general, it is assumed that the reservoir consists of CD4+ T cells that express no viral proteins. However, recent findings suggest that this may be an overly simplistic view and that the cells that contribute to the reservoir may be a diverse population that includes both CD4+ and CD4− cells. In this study, we directly infected resting CD4+ T cells and used fluorescence-activated cell sorting (FACS) and fiber-optic array scanning technology (FAST) to identify and image cells expressing HIV Gag. We found that Gag expression from integrated proviruses occurred in resting cells that lacked surface CD4, likely resulting from Nef- and Env-mediated receptor internalization. We also extended our approach to detect cells expressing HIV proteins in patients suppressed on ART. We found evidence that rare Gag+ cells persist during ART and that these cells are often negative for CD4. We propose that these double-negative α/β T cells that express HIV protein may be a component of the long-lived reservoir. IMPORTANCE A reservoir of infected cells persists in HIV-infected patients during antiretroviral therapy (ART) that leads to rebound of virus if treatment is stopped. In this study, we used flow cytometry and cell imaging to characterize protein expression in HIV-infected resting cells. HIV Gag protein can be directly detected in infected resting cells and occurs with simultaneous loss of CD4, consistent with the expression of additional viral proteins, such as Env and Nef. Gag+ CD4− cells can also be detected in suppressed patients, suggesting that a subset of infected cells express proteins during ART. Understanding the regulation of viral protein expression during ART will be key to designing effective strategies to eradicate HIV reservoirs.

Exploring Glycan Markers for Immunotyping and Precision-targeting of Breast Circulating Tumor Cells

BACKGROUND AND AIMS Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. METHODS We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FAST scan) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. RESULTS In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gp(C1). Using FAST-scan technology, we established a procedure to quantify expression levels of gp(C1) in tumor cells. In blood samples from five stage IV metastatic breast cancer patients, the gp(C1) positive CTCs were detected in all subjects; ∼40% of bCTCs were strongly gp(C1) positive. Interestingly, CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gp(C1) positive (92.5%, 37/40 CTCs). CONCLUSIONS Together we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gp(C1) as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients.

Abstract LB-344: MAP4K4 as a therapeutic target and novel biomarker for NSCLC

Introduction: Biomarker-guided personalized targeted therapies have improved lung cancer treatment; however, non-small cell lung cancer (NSCLC) targets such as KRAS — where activated mutations occur in ∼30% of cases — have so far eluded attempts at therapeutic targeting. There continues to be an urgent need to identify molecular mechanisms driving the disease and new targeted therapies. Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) overexpression promotes migration and invasion in several cancer types and correlates with poor prognosis in lung cancer. We investigated the role of MAP4K4 in NSCLC and tested whether MAP4K4 could serve as a biomarker on circulating tumor cells (CTCs) to monitor pre-metastatic and late stage disease. Methods: We used a novel class of highly selective small molecule inhibitor of MAP4K4, represented by SRI-28731, as a tool to further elucidate the role of MAP4K4 in NSCLC. We measured the expression of MAP4K4 and signaling changes following compound inhibition in lung cancer cell lines by immunoblotting and immunofluorescence. Clonogenic and wound healing assays were performed using our selective compound to assess proliferation and migration. To determine if MAP4K4 could be used as a biomarker in liquid biopsies we used FASTcell™, a non-enrichment optical scanning technology that enables identification and characterization of 1 tumor cell in 25 million blood cells. Results: We confirmed high expression of MAP4K4 in 4 out of 7 lung cancer cell lines, including A549 cells, and 3 of these harbored KRAS mutations. By clonogenic assay, A549 cell proliferation was inhibited by 51% and 99% with 2.5 nM and 5 nM of SRI-28731, respectively. In addition, lung cancer cell migration was impaired in the presence of 10 or 20 nM SRI-28731. Smad 1/5/8 phosphorylation was decreased in cells treated with the MAP4K4 inhibitor. Compound treated cells displayed a change in their actin cytoskeleton compared to DMSO treated cells. To explore whether MAP4K4 could be used as a biomarker on CTCs of metastatic NSCLC patients, we utilized the FASTcell™ platform and detected MAP4K4 expression on A549 cells mixed into normal blood. We are in the process of testing the assay with additional lung cancer cell lines and also testing MAP4K4 expression on CTCs from lung cancer patients. Conclusions: Our preliminary data suggest a role for MAP4K4 in proliferation, migration and signaling in lung cancer. Inhibition of MAP4K4 may be a valid therapeutic approach and the expression of MAP4K4 may serve as a biomarker in liquid biopsies. We showed that high levels of MAP4K4 in human lung cancer cell lines was concomitant to KRAS mutations, suggesting a possible correlation between the two markers. This novel biomarker could provide an opportunity to detect KRAS-positive cancers earlier and our new selective inhibitor could lead to life-saving, personalized treatment options for NSCLC cancer patients. Citation Format: Claire E. Repellin, Xiaohe Liu, Mary R. Stofega, Janey C.L. Snider, Zheng Ao, Ling Jong, Lidia C. Sambucetti. MAP4K4 as a therapeutic target and novel biomarker for NSCLC. [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 LB-344.

Abstract LB-C08: Exploring glycan markers for immunotyping and precision-targeting of breast circulating tumor cells (CTCs)

Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FASTcell) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gpC1. Using FASTcell technology, we established a procedure to quantify expression levels of gpC1 in tumor cells. In blood samples from five Stage IV metastatic breast cancer patients, the gpC1 positive CTCs were detected in all subjects; approximately 40% of bCTCs were strongly gpC1 positive. Interestingly, the CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gpC1 positive (92.5%, 37/40 CTCs). Taken together, we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gpC1 as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients. Citation Format: Lidia C. Sambucetti, Xiaohe Liu, Ben Hsieh, Richard Bruce, George Somlo, Jiaoti Huang, Denong Wang. Exploring glycan markers for immunotyping and precision-targeting of breast circulating tumor cells (CTCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C08.

Abstract 1205: In vivo animal models with patient-derived tumor xenografts for better clinical prediction

More predictive in vivo animal tumor models are urgently needed to test efficacy of newly developed cancer therapies and predict clinical response. At SRI we have developed highly innovative and predictive models initiated with single tumor cells isolated from cancer patient specimens, including colon, lung, and breast tumor xenografts. These tumors propagated in immunocompromised mice recapitulate primary tumor heterogeneity as shown by histology. In our models, the tumor cells are implanted subcutaneously to evaluate inhibition of primary tumor, or at orthotopic sites to recapitulate advanced metastatic disease and analyze circulating tumor cells (CTCs) or disseminated tumor cells (DTCs). Tumor cells are also implanted in organs, e.g. kidney capsule, to mimic the vascularized tumor microenvironment or in the human skin graft to analyze the tumor interactions with human stroma and vasculature, and the effect of treatment on angiogenesis. The cellular suspension method offers several advantages over the widely employed tumor fragments, including the ability to assess the phenotype of tumor cell populations, tumorigenicity, and tumor initiating cells. Cells isolated from tumors of mice in control and treatment groups are mouse-lineage depleted prior to evaluation in a number of in vitro or ex vivo assays, including migration and invasion through extracellular matrix proteins. To determine whether treatment affects frequency of tumor initiating cells (TICs) and metastasis initiating cells (MICs), we will perform in vivo limiting dilution assay (LDA). Moreover, to evaluate the ability of our tumor models to induce angiogenesis we utilized the chicken embryo chorioallantoic membrane (CAM) assay and scored the vascularity of mouse lineage-depleted tumor cells using an established CAM scoring guide with 5 as the highest score and 0 as the lowest score. One patient derived ER- invasive ductal carcinoma breast cancer (SRI-B1) with high growth rate demonstrated strong pro-angiogenic activity, inducing blood vessels in the CAM assay with a score of 4 compared to 5 for a VEGF positive control and 0 of negative control. The high vascular density induced by SRI-B1 tumor cells correlated with in vivo metastatic dissemination to distant organs upon orthotopic implantation in NOD/SCID mice, suggesting that tumor models can be screened in the CAM assay to evaluate their metastatic potential. We plan to further use these models to test the anticancer activity of experimental therapies that target metastasis. Citation Format: Jun Li, Jessica Kalra, Dominic Dinh, Wan-ru Chao, Xiaohe Liu, Lidia Sambucetti, Lucia Beviglia. In vivo animal models with patient-derived tumor xenografts for better clinical prediction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2014-1205

Abstract 731: SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy

MAP4K4, a Ser/Thr kinase, was identified as an important pro-migratory kinase in an siRNA screen, targeting 5,234 human genes for modulators of tumor cell motility. MAP4K4 siRNA potently suppressed cell invasion and migration of multiple cancer cell lines, indicating a broad role in cell motility. There are no drugs in the clinic that are known to specifically target MAP4K4 for cancer therapy. We have successfully developed an orally active, highly effective and selective MAP4K4 inhibitor (SRI-28731) with potent in vitro and in vivo anticancer activity. SRI-28731 is more potent than Paclitaxel (Taxol) against most of the breast cancer cell lines tested. SRI-28731 exhibits more potent activities against triple negative (MDA-MB-231, BT549 and Hs578T) than estrogen-dependent (T47D and MCF-7) breast cancer cell lines, and its potency is positively correlated with MAP4K4 expression in cancer cell lines. SRI-28731 is also more potent than Docetaxel against both androgen-dependent (LNCaP) and -independent (PC-3 and DU-145) prostate cancer cell lines. In vitro mechanistic studies showed that SRI-28731 induced apoptosis and a time-dependent M phase arrest. Treatment with SRI-28731 (12.5, 25 and 50 mg/kg/day) caused a significant dose-dependent growth reduction of PC-3 tumors (30%, 61% and 88% growth inhibition, respectively), while Docetaxel at its MTD (7.5 mg/kg; Q3Dx2) produced only 10-15% growth inhibition. At the end of PC-3 tumor xenograft studies, we conducted an ex vivo invasion assay using PC-3 tumor cells isolated from tumor-bearing mice. SRI-28731 significantly reduced ex vivo tumor cell invasion by ∼80%. Pharmacokinetic studies showed that SRI-28731 could be detected in plasma up to 8 hours after oral dosing, and drug plasma concentrations remained above the IC50 values needed to inhibit prostate or breast cancer proliferation. To quantitatively define the kinase selectivity of SRI-28731, we tested the interaction of SRI-28731 with 456 kinase protein kinases (KINOMEscan), followed by in vitro pharmacology studies. Our data indicated that SRI-28731 is a highly selective Type-II MAP4K4 inhibitor. Type II kinase inhibitors bind to both the ATP site and an adjacent hydrophobic site exposed in the non-activated kinase state. Generally, type II inhibitors show higher selectivity for targets, and act primarily by locking the equilibrium switch between conformational states in a way that prevents kinase activation, rather than directly inhibiting it. Elevated MAP4K4 expression is strongly associated with higher rate of metastasis, and is regarded as an independent predictor of overall survival in cancer patients. Since MAP4K4 is overexpressed in many human cancer cell lines but is undetectable in non-transformed epithelial cells, targeting MAP4K4 may provide effective anti-metastatic therapy with limited side effects on normal tissues. Citation Format: Chih-Tsung Chang, Jaehyeon Park, Wei Zhou, Xiaohe Liu, Barbara Sato, Dominic Dinh, Anna Furimsky, Lucia Beviglia, Lidia Sambucetti, Ling Jong. SRI-28731, a highly potent and selective MAP4K4 (HGK) inhibitor for cancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 731. doi:10.1158/1538-7445.AM2014-731

Abstract A199: Using Fiber Array Scanning Technology (FAST) Platform for sensitive detection and analysis of circulating tumor cells with a hybrid epithelial/mesenchymal phenotype from non-small cell lung cancer.

Circulating tumor cell (CTC) analysis uses a “liquid biopsy” to provide critical information regarding diagnosis, prognosis, response to therapy and risk of relapse. To date, CTC analysis in non-small cell lung cancer (NSCLC) patients using epithelial cell adhesion molecule (EpCAM) immunomagnetic enrichment techniques has met with limited success. This is thought to be because the majority of CTCs detected in NSCLC patients demonstrate the mesenchymal phenotype to various extents, and epithelial markers on CTCs are lost during the epithelial-mesenchymal transition (EMT). We report here that the non-enrichment, FAST platform can be used for rapid detection and characterization of CTCs with a hybrid EMT phenotype. We developed a model using whole blood from healthy donors spiked with human NSCLC cell lines that have been classified as having varying levels of EMT. These included “more epithelial, high EpCAM type” (A549 and EKVX), and “more mesenchymal, low EpCAM type” (H2228 and Calu-1) cell lines. A multiplexed assay was developed to simultaneously evaluate cellular and molecular markers of the hybrid phenotypes. FAST was used to develop an optimized assay with high yields (more than 75% retention, detection performance as high as 1 CTC/5 ml blood) of CTCs with hybrid phenotype. 100% of NSCLC cells detected by the FAST platform co-expressed mesenchymal/stem cell markers such as Vimentin, N-Cadherin and ALDH1. FACS analysis of NSCLC cells confirmed levels of EpCAM expression ranging from negative to high. CTCs harboring anaplastic lymphoma kinase (ALK) translocations also demonstrated mesenchymal-like phenotypes. Finally, a high incidence of CTC clusters (circulating tumor microemboli; CTM) that have been previously reported with the hybrid phenotype, was detected from Stage IV NSCLC patients. These data illustrate that the FAST platform is suitable for high-sensitivity detection and comprehensive analyses of the full spectrum of EMT phenotypes in NSCLC CTCs. We have also developed assays to multiplex cellular and molecular biomarkers on single CTCs to guide personalized therapy, including the detection of ALK to select patients appropriate for Crizotinib therapy. Further work is planned to analyze the dynamic change of EMT components in CTCs during disease progression to correlate EMT biomarkers in CTCs with patient outcomes and provide new diagnostic tools for lung cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A199. Citation Format: Xiaohe Liu, Janey C. Ly, Laurie Kara, Heather Wakelee, Nathan Collins, Keith Laderoute, Lidia C. Sambucetti. Using Fiber Array Scanning Technology (FAST) Platform for sensitive detection and analysis of circulating tumor cells with a hybrid epithelial/mesenchymal phenotype from non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A199.

Abstract 2146: Development of functional assays for p97/VCP inhibition.

p97 (also called VCP in metazoans and CDC48 in yeast) is a highly conserved, ubiquitously expressed, and essential AAA ATPase. p97 plays a key role in endoplasmic reticulum-associated degradation of misfolded secretory and membrane proteins as well as ubiquitin-dependent turnover of a subset of cytoplasmic substrates of the ubiquitin-proteasome system.

Abstract 1692: SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC SR16388 is a novel amino steroid that targets estrogen-binding proteins including genomic estrogen receptors (ERs). Preclinical studies have demonstrated that SR16388 is a well-tolerated, orally active compound with compelling properties as an experimental antitumor agent: SR16388 has been demonstrated to 1) inhibit the proliferation and viability of diverse human cancer cell lines in vitro; 2) have antiangiogenic activity in vivo; and 3) reduce the growth of human tumor xenografts in mice. SR16388, which binds to and potently inhibits ERα and ERβ in breast cancer cells, also inhibits the widely expressed orphan nuclear receptor ERRα that has been implicated in the development of various human malignancies. ERRα is thought to regulate tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. We have been exploring potential mechanisms that could explain SR16388’s broad anti-proliferative effects toward human tumor cell lines with different genotypes and tissues of origin. In these studies, we observed that SR16388 can initiate a cell-cycle arrest at G1 or G2 and ultimately cause cytotoxicity (e.g., by apoptosis) even in cells that do not express ERs or ERRα. We recently observed that SR16388 can induce persistent macroautophagy (autophagy) in diverse tumor cells, including cells from hematopoietic malignancies (e.g., multiple myeloma/MM cells) in addition to carcinomas such as prostate and breast cancers. We demonstrated induction of autophagy by using established assays (e.g., LC3I/II processing; fluorescent reporters for acidic vacuole formation; long-lived protein degradation). Here we present results demonstrating that SR16388 can rapidly and potently induce autophagy in cultures of MM cells (U266, RPMI-8226) and PC3 prostate cancer cells. Induction of autophagy in PC3 cells, in particular, was detected in tumor xenografts as early as 3 hours after dosing of mice with a therapeutic dose level of SR16388. We will present data supporting the hypothesis that SR16388 exerts its anti-proliferative and anti-tumor effects in part through the induction of stress pathways modulated by perturbations of protein homeostasis. These effects could yield therapeutic benefits that derive from targets within solid tumor microenvironments. Citation Format: Lidia C. Sambucetti, Wei Zhou, Joy Calaoagan, Laurie Kara, Xiaohe Liu, Barbara Sato, Steve Samuelson, Nathan Collins, Keith Laderoute. SR16388 impairs protein homeostasis and induces autophagy in diverse human cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1692. doi:10.1158/1538-7445.AM2013-1692