The morphologies of breast cancer cell lines in three-dimensionalassays correlate with their profiles of gene expression

3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments

A Method to Find Longevity-Selected Positions in the Mammalian Proteome

Evolutionary theory suggests that the force of natural selection decreases with age. To explore the extent to which this prediction directly affects protein structure and function, we used multiple regression to find longevity-selected positions, defined as the columns of a sequence alignment conserved in long-lived but not short-lived mammal species. We analyzed 7,590 orthologous protein families in 33 mammalian species, accounting for body mass, phylogeny, and species-specific mutation rate. Overall, we found that the number of longevity-selected positions in the mammalian proteome is much higher than would be expected by chance. Further, these positions are enriched in domains of several proteins that interact with one another in inflammation and other aging-related processes, as well as in organismal development. We present as an example the kinase domain of anti-Müllerian hormone type-2 receptor (AMHR2). AMHR2 inhibits ovarian follicle recruitment and growth, and a homology model of the kinase domain shows that its longevity-selected positions cluster near a SNP associated with delayed human menopause. Distinct from its canonical role in development, this region of AMHR2 may function to regulate the protein’s activity in a lifespan-specific manner

A human breast cell model of pre-invasive to invasive transition

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from pre-invasive to invasive phenotype as it may occur 'spontaneously' in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted-basement membrane cultures. These cells remained non-invasive; however, unlike their non-malignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher grade tumors. To find functionally significant changes in transition from pre-invasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between pre-invasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP-9,-13,-15,-17 was up regulated in the invasive cells. Using siRNA based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which pre-invasive breast cells could acquire invasiveness in a metaplastic context

On Two Problems in Comparative Genomics of Eukaryotes

The recent advent of whole-genome sequencing allows us to use novel comparative methods to explore the genetic bases for traits of interest. Here, I present two case studies of such methods applied to eukaryote genomes. The first study regards the evolution of longevity in the mammalian proteome. Evolutionary theory suggests that the force of natural selection decreases with age. To explore the extent to which this prediction directly affects protein structure and function, I used computational methods to identify positions of proteins conserved in long-lived but not in short-lived mammal species. I analyzed 7,590 orthologous protein families in 33 mammalian species, accounting for body mass, phylogeny, and species-specific mutation rate. Overall, I found that the number of longevity-selected positions in the mammalian proteome is much greater than would be expected by chance. Further, these positions are enriched in domains of several proteins that interact with one another in inflammation and other aging-related processes, as well as in organismal development. I present as an example the kinase domain of anti-Müllerian hormone type-2 receptor (AMHR2). AMHR2 inhibits ovarian follicle recruitment and growth, and my results show that its longevity-selected positions cluster near a SNP associated with delayed human menopause. Distinct from its canonical role in development, this region of AMHR2 may function to regulate the protein's activity in a lifespan-specific manner. The second study concerns the genetic basis for toxin production in the black mold genus Stachybotrys, which produces several diverse toxins that can damage human health. Its strains comprise two mutually-exclusive toxin chemotypes, one producing satratoxins (a subclass of trichothecenes) and the other producing the less-toxic atranones. To determine the genetic bases for chemotype-specific differences in toxin production, I sequenced and assembled de novo four Stachybotrys genomes, including two from atranone strains and two from satratoxin strains. Comparative analysis of these four 35-Mbp genomes revealed several chemotype-specific gene clusters that are predicted to make atranones and satratoxins, based on several lines of evidence. I show that chemotype-specific gene clusters are likely the genetic basis for the mutually-exclusive toxin chemotypes of Stachybotrys. I then present a unified biochemical model for Stachybotrys toxin production

Evaluating Differences in Patient Perception of Effectiveness of Oral Versus Parenteral Non-Steroidal Anti-Inflammatory Drugs

The 53rd Annual Medical Student Research Forum at UT Southwestern Medical Center (Monday, January 26, 2015, 2-5 p.m., D1.602)BACKGROUND: Pain is a very common reason for emergency department (ED) visits. Providing safe, effective analgesia is not only central to providing good health care, but also to overall patient satisfaction. Prior work has shown that although parenteral administration of NSAIDs is more expensive and has greater risk of adverse effects than oral administration, there are no significant differences between the two routes in amount or efficiency of pain relief. However, some clinicians believe that patients presenting with pain to the ED prefer parenteral over oral NSAIDs, due to an assumed patient perception that shots are more effective than pills. Additionally, prior studies in this area have not examined patient ethnicity as a factor in administration preference. OBJECTIVE: To determine the extent to which patient preference for oral versus parenteral methods of NSAID administration can be influenced by education. METHODS: All patients who presented to a county hospital ED with a chief complaint of pain and were subsequently prescribed any type of NSAID were included. After obtaining consent, a survey was given in English or Spanish to obtain demographic data and question the patient on three items: (1) Which NSAID form (pill or shot) is better at taking away pain (2) Which NSAID form is faster at taking away pain (3) Which NSAID form is preferred to take away pain. This preference survey was repeated after educating the patient that the shot and the pill are equally effective but the shot costs more and causes pain. Statistical analysis was performed using standard methods. RESULTS: N=270 patients were surveyed, with mean age 42; 49% female; and 37% African American, 37% Hispanic, and 23% Caucasian. Before education, overall subjects were equally likely to prefer the pill versus the shot but believed the shot to be faster (82%) than the pill (17%). Patient education on NSAIDs significantly influenced patient beliefs on all three items: 73% found the pill to be as effective as the shot, 55% found the pill to be as fast as the shot, and 75% preferred the pill (p<0.001 in all three cases). There were no statistically significant differences in any of these beliefs across patient ethnicity, education, or narcotic preference. CONCLUSION: Despite clinician assumptions, most patients have favorable attitudes toward NSAID pill versus shot effectiveness and overall preference, and can be easily influenced by briefly educating these patients in the ED. This suggests brief patient education is effective to reduce cost of treating minor pain in the ED. Future work should compare effectiveness of various education methods.Southwestern Medical Foundatio

Conceptual example of our multiple regression method applied to a single column (Y465 of AMHR2; full result in Table S2).

<p>(left) Characters shown ordered by species MLS. For each non-human species, we calculate the similarity score (";BLOSUM80";) for the species’ amino acid character versus the human character (here Y); e.g., this score for Tursiops would be the similarity score for H versus Y, which is 2. (right) We then fit the MLS of all non-human species to their similarity scores; e.g., Tursiops’ contribution to this fit is the point (52, 2). Not shown are the steps to correct for mutation rate and shared phylogeny, and the simultaneous fit of body mass. For this column, the data provide relatively strong support for a nonzero slope in the fit of similarity to MLS, even given trends in mutation rate, phylogeny, and body mass, and so this position is assigned a relatively significant p-value (<i>p_MLS</i><0.01).</p

Predicted secondary structure in all positions of the human proteome and in two subsets of longevity-selected positions.

<p>Predicted secondary structure in all positions of the human proteome and in two subsets of longevity-selected positions.</p

OrthoMaM alignment of the C-terminal regions of AMHR2 ortholog kinase domains.

<p>Orthologs are ordered by species MLS. The five longevity-selected positions in this region (Y465, T469, F473, E513, and H515) are highlighted in gray. ";X"; indicates regions that we masked due to excessive divergence (Section 2.1). Long regions of gaps are not necessarily real genome deletions, but are more likely to have been missed during genome assembly or annotation.</p

Density histograms of <i>p_MLS</i> values.

<p>A. Real data. B. Randomized control. Each histogram shows the n=734,741 fit positions with <i>b_MLS</i>>0. See Section 3.2 for details.</p

AMHR2 kinase domain mapped onto experimental structure of BMPR2 kinase domain.

<p>All eight longevity-selected positions found in this domain are shown as black sticks and are further described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038595#pone.0038595.s003" target="_blank">Table S2</a>. The five longevity-selected positions discussed in the text are labeled; they are found on the αG–αH loop (Y465, T469, and F473) and the C-terminal loop following αI (E513 and H515).</p