Larry C. Cheng

Dietary γ-Tocopherol–Rich Mixture Inhibits Estrogen-Induced Mammary Tumorigenesis by Modulating Estrogen Metabolism, Antioxidant Response, and PPARγ

This study evaluated the anticancer activity and mechanism of action of a γ-tocopherol–rich tocopherol mixture, γ-TmT, in two different animal models of estrogen-induced breast cancer. The chemopreventive effect of γ-TmT at early (6 weeks), intermediate (18 weeks), and late (31 weeks) stages of mammary tumorigenesis was determined using the August-Copenhagen Irish rat model. Female rats receiving 17β-estradiol (E2) implants were administered with different doses (0%, 0.05%, 0.1%, 0.3%, and 0.5%) of γ-TmT diet. Treatment with 0.3% and 0.5% γ-TmT decreased tumor volume and multiplicity. At 31 weeks, serum concentrations of E2 were significantly decreased by γ-TmT. γ-TmT preferentially induced expression of the E2-metabolizing enzyme CYP1A1, over CYP1B1 in the rat mammary tissues. Nrf2-dependent antioxidant response was stimulated by γ-TmT, as evident from enhanced expression of its downstream targets, NQO1, GCLM, and HMOX1. Serum concentrations of the oxidative stress marker, 8-isoprostane, were also decreased in the γ-TmT–treated groups. Treatment with γ-TmT increased expression of PPARγ and its downstream genes, PTEN and p27, whereas the cell proliferation marker, PCNA, was significantly reduced in γ-TmT–treated mammary tumors. In an orthotopic model in which human MCF-7 breast cancer cells were injected into the mammary fat pad of immunodeficient mice, γ-TmT inhibited E2-dependent tumor growth at all the doses tested. In conclusion, γ-TmT reduced mammary tumor development, in part through decreased E2 availability and reduced oxidative stress in mammary tissues; γ-TmT could thus be an effective agent for the prevention and treatment of E2-induced breast cancer. Cancer Prev Res; 8(9); 807–16. ©2015 AACR.

Integrating phosphoproteomics into the clinical management of prostate cancer

Phosphoproteomic analysis of tumor samples has the potential to uncover significant insights into kinase signaling networks present in late stage prostate cancer that are complementary to genomic and transcriptomic approaches. Phosphoproteomics could potentially aid drug development in clinical trial design as well as provide utility for oncologists in the personalized therapeutic management of individual cancers through identifying novel biomarkers and druggable targets. Rapid advancement of targeted mass spectrometry platforms is underway to integrate phosphoproteomic technology with genomic assays to soon translate this information into the cancer clinic.

Complementing genomics and transcriptomics: Phosphoproteomics illuminates systems biology in prostate cancer.

ABSTRACT Integration of phosphoproteomics with traditional genomics and transcriptomics provides a more comprehensive overview of the signaling networks in advanced prostate cancer for immediate preclinical and future clinical use. Our recent publication introduces computational approaches for integrating the phosphoproteome, specifically with the intent of identifying important kinase signaling networks in advanced-stage prostate cancer.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

Abstract 1912: Reduction of microRNA-21 by vitamin D compounds during ductal carcinoma in situ transition to invasive ductal carcinoma

MicroRNAs (miRNAs) are small RNA molecules of approximately 22 nucleotides in lengths that can negatively control target gene expression post-transcriptionally. There are currently more than one hundred known human miRNAs. miRNAs have been shown to play an important role in the development and progression of various types of cancer. In breast cancer, ductal carcinoma in situ (DCIS) is an early non-invasive precursor, which is known to progress to invasive ductal carcinoma (IDC) if left untreated. The involvement of miRNAs in the breast cancer progression from DCIS to IDC is still not well understood, and potential regulation of miRNAs by vitamin D compounds during the progression has not been tested. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), inhibits the progression of DCIS to IDC in vivo. To identify miRNAs that could potentially be involved in DCIS progression to IDC, we utilized the MCF10DCIS.com xenograft tumors. MCF10DCIS.com cells (10^6 cells/mouse) were injected subcutaneously into the rear flanks of nu/nu mice. The mice were then treated with BXL0124 (0.1 μg/kg) for 5 weeks. The critical DCIS to IDC transition occurred between weeks 3 and 4 in the in vivo lesions, and several microRNAs were assessed with respect to the DCIS to IDC transition at weeks 3 and 4. Among many miRNAs analyzed, miR-21, miR-24, and miR-140 were up-regulated in IDC lesions compared to DCIS lesions, whereas no marked changes were observed with respect to miR-200c, miR-let-7a, miR-26a, miR-34a, and miR-182. One of the miRNAs identified miR-21, is a well-known oncomir and overexpressed in breast carcinoma, mediating cell survival, proliferation, and metastasis. The increased expression of miR-21 was significant when DCIS lesions progressed to IDC, and this effect was diminished by the treatment with Gemini vitamin D, BXL0124, at week 4. In conclusion, vitamin D compounds reduce the levels of miR-21 during DCIS transition to IDC, potentially contributing to its preventive effects in breast cancer progression. Further investigation of the mechanisms of action by which vitamin D compounds regulate miR-21 should be considered. (This work was supported in part by the National Institutes of Health National Cancer Institute R01-CA127645 and the National Institute of Environmental Health Sciences Grant ES005022). Citation Format: Joseph Wahler, Larry C. Cheng, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. Reduction of microRNA-21 by vitamin D compounds during ductal carcinoma in situ transition to invasive ductal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1912. doi:10.1158/1538-7445.AM2015-1912

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

MAPK reliance via acquired CDK4/6 inhibitor resistance in cancer

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1–S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon. Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer. Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor–resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion. Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201–14. ©2018 AACR.

Microarray therapeutic index: A novel method to combine molecular and clinical data from cancer patients

A new method has been developed that enables combining the microarray and clinical data (e.g., survival months) of cancer patients in order to calculate what is here referred to as the individualized therapeutic index (ITI). The prerequisite for calculating ITI includes (i) the measurement of the microarray (or micro) therapeutic index, mTI, defined here for the first time, and (ii) the conversion of mTI into ITI through filtering off those genes whose transcript dynamics cause deviations of points from the regression line of the survival months vs. mTI (SmTI) plot. The ability to measure ITI values of drugs opens up new opportunities to improve drug discovery research (i.e., theragnostics) and personalized medicine.