Joel A. Yates

Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8

MINT‐7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)

Regulation of Androgen-Responsive Transcription by the Chromatin Remodeling Factor CHD8

The androgen receptor (AR) mediates the effect of androgens through its transcriptional function during both normal prostate development and in the emergence and progression of prostate cancer. AR is known to assemble coactivator complexes at target promoters to facilitate transcriptional activation in response to androgens. Here we identify the ATP-dependent chromatin remodeling factor chromodomain helicase DNA-binding protein 8 (CHD8) as a novel coregulator of androgen-responsive transcription. We demonstrate that CHD8 directly associates with AR and that CHD8 and AR simultaneously localize to the TMPRSS2 enhancer after androgen treatment. In the LNCaP cell line, reduction of CHD8 levels by small interfering RNA treatment severely diminishes androgen-dependent activation of the TMPRSS2 gene. We demonstrate that the recruitment of AR to the TMPRSS2 promoter in response to androgen treatment requires CHD8. Finally, CHD8 facilitates androgen-stimulated proliferation of LNCaP cells, emphasizing the physiological importance of CHD8. Taken together, we present evidence of a functional role for CHD8 in AR-mediated transcriptional regulation of target genes.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including 13C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Macrophages Enhance Migration in Inflammatory Breast Cancer Cells via RhoC GTPase Signaling

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC’s extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC’s hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM “primes” the IBC cells’ cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins −6, −8, and −10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

ABSTRACT Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P3 resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived ‘signaling-capable’ CCPs. Highlighted Article: EGFR and PTEN localize to short-lived clathrin-coated pits (CCPs), and PTEN regulates CCP dynamics and signaling, suggesting compartmentalized signaling is crucial for proper signaling transduction.

Loss Of PTEN Promotes Formation Of Signaling-Specific Clathrin-Coated Pits

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived ‘signaling-capable’ CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

Abstract 1040: Differential levels of glycogen in breast cancer cell lines: A potential new target

Cancer cells have been known to alter their metabolic processes in order to survive and proliferate. Normally in muscle and liver, excess glucose is stored within the cells as glycogen. Elevated levels of glycogen have also been found in various cancers, including breast cancers. Recent studies have implicated glycogen metabolism as important in promoting survival of cancer cells, suggesting targeting of glycogen metabolism as a possible treatment to inhibit cancer cell growth. In general, modulation of cancer metabolism is believed to be an attractive adjunct strategy to conventional or targeted therapies. Here we set out to investigate glycogen levels as well as levels of proteins involved in glycogen synthesis and degradation vary across different breast cancer cell lines. A glucose metabolism qPCR array found differential levels of the alpha subunit of phosphorylase kinase 1, a key enzyme involved in glycogen degradation among three different breast cancer cell lines. Expression levels of glycogen synthesis and degradation enzymes were assessed using qPCR and immunoblot in various breast cancer cell lines. Glycogen levels in these breast cancer cell lines were quantified using an amyloglucosidase reaction coupled with other enzymatic reactions to produce a fluorescent product. It was found that MDA-MB-231, SUM149, and MCF7 cell lines had increased levels of glycogen, between 6.5 and 23.5 μg glycogen per mg protein, whereas SUM190 and normal-like breast epithelial cell line MCF10A had undetectable levels of glycogen. These findings demonstrate that glycogen metabolism can vary widely amongst cancer types, indicating that therapies targeted to disrupt glycogen degradation may produce differential results and that further study of the role of glycogen metabolism in cancer is warranted. Citation Format: Joel A. Yates, Megan Altemus, Zhifen Wu, Michelle L. Wynn, Sofia D. Merajver. Differential levels of glycogen in breast cancer cell lines: A potential new target. [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 1040.

Abstract 1203: Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149

Metabolic reprogramming is increasingly recognized as a fundamental hallmark of cancer. While the Warburg effect and normal proliferative metabolism are similar, they are not equivalent. We hypothesize that there are key drivers of malignant metabolism that can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Using 13C-labeled glucose and glutamine tracers in combination with mass spectrometry and measurements of extracellular glucose, lactate, and glutamine flux, we have characterized system level differences in a series of breast cancer cell lines as well as normal-like breast epithelial cells. We observed an increase in the reductive carboxylation of glutamine-derived citrate and alpha-ketoglutarate in the triple-negative inflammatory breast cancer cell line SUM149. We also observed that the SUM149 exhibit high levels of HIF-1α and low levels of oxygen consumption under normoxia, suggesting that the cell line is highly adapted to hypoxia. Surprisingly, the stable depletion of HIF-1α via shRNA had no significant effect on the metabolic profile of these cells. Previous work by our lab and others has demonstrated that the GTPase RhoC is a driver of the metastatic phenotype exhibited by inflammatory breast cancer. Activation of RhoC is known to induce cytoskeletal rearrangements and increase invasive potential. The Rho GTPase family of proteins has also recently been linked to metabolism, specifically regulation of glutaminase activity. Here we show that stable knockdown of RhoC in SUM149 cells results in a marked decrease in the rate of both glutamine uptake and intracellular reductive carboxylation. This work reinforces the role of RhoC as an important driver of inflammatory breast cancer metastatic potential. We conclude that RhoC remains an important clinical target with the potential to alter patient outcomes. Citation Format: Joel A. Yates, Michelle L. Wynn, ZhiFen Wu, Charles R. Evans, Charles Burant, Santiago D. Schnell, Sofia D. Merajver. Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149. [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 1203. doi:10.1158/1538-7445.AM2015-1203