Cecil J. Gomes

Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells.

Obesity and a western diet have been linked to high levels of bile acids and the development of colon cancer. Specifically, increased levels of the bile acid deoxycholic acid (DCA), an established tumor promoter, has been shown to correlate with increased development of colorectal adenomas and progression to carcinoma. Herein we investigate the mechanism by which DCA leads to EGFR-MAPK activation, a candidate mechanism by which DCA may promote colorectal tumorigenesis. DCA treated colon cancer cells exhibited strong and prolonged activation of ERK1/2 when compared to EGF treatment alone. We also showed that DCA treatment prevents EGFR degradation as opposed to the canonical EGFR recycling observed with EGF treatment. Moreover, the combination of DCA and EGF treatment displayed synergistic activity, suggesting DCA activates MAPK signaling in a non-canonical manner. Further evaluation showed that DCA treatment increased intracellular calcium levels and CAMKII phosphorylation, and that blocking calcium with BAPTA-AM abrogated MAPK activation induced by DCA, but not by EGF. Finally we showed that DCA-induced CAMKII leads to MAPK activation through the recruitment of c-Src. Taken together, we demonstrated that DCA regulates MAPK activation through calcium signaling, an alternative mechanism not previously recognized in human colon cancer cells. Importantly, this mechanism allows for EGFR to escape degradation and thus achieve a constitutively active state, which may explain its tumor promoting effects.

The induction of endoreduplication and polyploidy by elevated expression of 14-3-3γ

Several studies have demonstrated that specific 14-3-3 isoforms are frequently elevated in cancer and that these proteins play a role in human tumorigenesis. 14-3-3γ, an isoform recently demonstrated to function as an oncoprotein, is overexpressed in a variety of human cancers; however, its role in promoting tumorigenesis remains unclear. We previously reported that overexpression of 14-3-3γ caused the appearance of polyploid cells, a phenotype demonstrated to have profound tumor promoting properties. Here we examined the mechanism driving 14-3-3γ-induced polyploidization and the effect this has on genomic stability. Using FUCCI probes we showed that these polyploid cells appeared when diploid cells failed to enter mitosis and subsequently underwent endoreduplication. We then demonstrated that 14-3-3γ-induced polyploid cells experience significant chromosomal segregation errors during mitosis and observed that some of these cells stably propagate as tetraploids when isolated cells were expanded into stable cultures. These data lead us to conclude that overexpression of the 14-3-3γ promotes endoreduplication. We further investigated the role of 14-3-3γ in human NSCLC samples and found that its expression is significantly elevated in polyploid tumors. Collectively, these results suggests that 14-3-3γ may promote tumorigenesis through the production of a genetically unstable polyploid intermediate.

Measuring DNA content in live cells by fluorescence microscopy

BackgroundLive-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell’s integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique.ResultsPresented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm’s accurate assessment of DNA content was validated by parallel flow cytometric studies.ConclusionsThis method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

Abstract 713: Determining the role of tumor-infiltrating B cells in NSCLC

Recent studies in human non-small cell lung cancer (NSCLC) have shown that upregulation of B cell-associated genes strongly correlate with early stage patient survival. After analyzing a gene expression database of lung tumors we showed that CD79A, a pan-B cell marker, had the strongest predictive value, suggesting that B cells are playing a crucial role in NSCLC immunity. In order to evaluate this we first examined where this genetic signature was originating from. We observed that patients with high levels of B cell-related genes also showed high numbers of CD79A+ B cells intimately associated with the tumor, but not being expressed by tumor cells themselves. A closer look at tumor-infiltrating B cells (TIL-B cells) by IHC and flow cytometry of fresh patient tumor samples has confirmed their presence and allowed us to begin elucidating their phenotype. Interestingly, we have found that not all early stage patients have detectable levels of TIL-B cells, but in those that do, TIL-B cells represent a marked proportion of the lymphocytic infiltration. Further evaluation of T cell populations in the same samples indicate that T cell numbers remain relatively consistent but that the numbers of B-cells, especially CD79A+ B-cells, does vary from patient to patient. These data indicate that CD79A+ TIL-B cells are contributing to the generation of an efficient immune response in some but not all patients, and are greatly influencing disease survivability. This information may allow us to stratify patients into low and high risk groups based on the presence or absence of TIL-B cells, respectively. Citation Format: Sara M. Centuori, Samuel Kim, Cecil Gomes, Charles Putnam, David Mount, Linda Garland, Brandon Larsen, Jesse D. Martinez. Determining the role of tumor-infiltrating B cells in 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 713.

Abstract 4329: The consequence of obesity and obstructive sleep apnea in liver cancer

Primary liver cancer is the 3rd leading cause of cancer mortality worldwide. Eighty-five percent of liver cancers is comprised of hepatocellular carcinoma (HCC). Risk of developing HCC increases when patients have obesity related comorbidities, involving non-alcoholic steatohepatitis (NASH), hepatic inflammation, or lipid accumulation. Additionally, obesity is generally regarded as a prime risk factor for the development of obstructive sleep apnea (OSA), a repeated obstruction of the upper airway during sleep leading to oxygen deficiency (OD), which also increases the incidence of NASH. The connection of these morbidities presume that patients suffering from obesity induced OSA have an increased risk of developing HCC. To examine obesity and OSA9s role in HCC we combined three generally accepted methods: A one-time injection of tumor initiator diethylnitrosamine followed by continuous administration of the promoter phenobarbital, a high fat diet, and a hypoxia chamber to house the mice for regulation of their oxygen intake, allowing us to mimic the OD seen in OSA. Utilizing these treatment methods and applying microCT imaging to non-invasively and repeatedly monitor the liver, we have been able to examine the effects of obesity and/or OD. Preliminary result indicate that a high fat diet results in obesity, lipidosis and liver damage. Interestingly, OD seems to inhibit obesity while still generating hepatic inflammation. Furthermore, we have observed that obesity and OD together cause severe damage to the liver, enhancing the risk of developing HCC. This study will be a significant preclinical step in distinguishing mechanisms that may explain obesities and OSA9s combined consequence in HCC. Citation Format: Nathan W. Sweeney, Cecil J. Gomes, Sairam Pathasarthasy, Jesse D. Martinez. The consequence of obesity and obstructive sleep apnea in liver 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 4329.

Abstract 3591: Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells

In normal lung tissue the expression levels of 14-3-3 gamma are very low and tightly regulated, however, in cancer it is evident that 14-3-3 gamma9s expression patterns become deregulated and significantly elevated. In patients with advanced non-small cell lung cancer (NSCLC), increased expression of this isoform is associated with poorer survival and significantly correlates with lymph node and distant metastasis. These data prompted us to analyze TCGA9s NSCLC gene expression dataset, and it is clear that 14-3-3 gamma9s expression continues to increase in the progression from early to late stage cancers. Taken together, these data suggest that overexpression of 14-3-3 gamma is correlated with a more aggressive tumor phenotype, an observation also seen with breast and hepatocellular carcinoma. The focus of this study is to elucidate the mechanism(s) causing these more aggressive cancer phenotypes. We have previously shown that overexpressing 14-3-3 gamma in human lung cancer cells harboring very low levels of endogenous 14-3-3 gamma and no wildtype p53 results in a stable subpopulation of cells with polyploid DNA content. Interestingly, approximately 40% of lung adenocarcinomas present with hyper-triploid karyotypes, and even a higher percentage, 40-60%, have inactivation of p53. It is well established that polyploid tumors have the capacity to increase tumorigenicity by allowing resistance to conventional therapies and also permitting elevated tolerance to chromosomal instability (CIN). This leads us to hypothesize that in the absence of p53, NSCLC tumors overexpressing 14-3-3 gamma result in a polyploid population of cells that may be influencing the aggressiveness of the tumor. Further in vitro analysis of these 14-3-3 gamma induced tetraploid cells show that they have a prolonged mitosis with significantly more lagging chromosomes in anaphase than their diploid counterparts, indicating an increase in chromosomal instability (CIN). After isolating isogenic tetraploid clones with or without 14-3-3 gamma expression, the stability of the tetraploid cell state was assessed. Clones not expressing 14-3-3 gamma quickly reverted back to a pseudo-diploid state, whereas overexpression of 14-3-3 gamma significantly prolonged the tolerance of tetraploidy and eventually resulted in an aneuploid cell state. Our data suggests that overexpression of 14-3-3 gamma increases tumorigenicity through the stabilization of a polyploid cell state. Citation Format: Cecil J. Gomes, Michael Harman, Sara Centuori, Charles Wolgemuth, Jesse Martinez. Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells. [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 3591.

Abstract 3509: Overexpression of 14-3-3γ contributes to chromosomal instability in human lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Lung cancer is the most common cause of cancer-related deaths, and nearly all lung cancers exhibit genomic instability which results in an abnormal number of chromosomes known as aneuploidy. In many lung cancers, 14-3-3 proteins have been shown to be aberrantly activated or suppressed, which implies that these proteins have potential roles in tumorigenesis. Our lab has demonstrated that over expression of one 14-3-3 family member, 14-3-3γ, in a non-small cell lung cancer cell line leads to an increase in the number of cells that exhibit polyploidy, suggesting that this 14-3-3 protein can disrupt normal chromosome segregation. Additionally, overexpression of 14-3-3γ leads to atypical DNA replication and cell cycle progression. Further evaluation of the polyploid population induced by overexpressing 14-3-3γ revealed that a majority of the cells were multinucleated, often with two or more nuclei of unequal size and morphology. Live cell microscopy shows that this nonlinear marked increase in nuclear content is due to unequal distribution of DNA to the daughter cells upon cellular division, promoting chromosomal instability. Our data indicates that 14-3-3γ may play an important role in maintaining normal diploidy, but when overexpressed, as seen in many lung cancers, can lead to increased levels of chromosomal instability and progression into aneuploidy. Citation Format: Cecil J. Gomes, Sara Centuori, Jesse D. Martinez. Overexpression of 14-3-3γ contributes to chromosomal instability in human lung cancer. [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 3509. doi:10.1158/1538-7445.AM2014-3509