Patrick B. Thomas

A longitudinal study of the impact of chronic psychological stress on health-related quality of life and clinical biomarkers: protocol for the Australian Healthy Aging of Women Study

BackgroundDespite advancements in our understanding of the importance of stress reduction in achieving good health, we still only have limited insight into the impact of stress on cellular function. Recent studies have suggested that exposure to prolonged psychological stress may alter an individual’s physiological responses, and contribute to morbidity and mortality. This paper presents an overview of the study protocol we are using to examine the impact of life stressors on lifestyle factors, health-related quality of life and novel and established biomarkers of stress in midlife and older Australian women.The primary aim of this study is to explore the links between chronic psychological stress on both subjective and objective health markers in midlife and older Australian women. The study examines the extent to which exposure frightening, upsetting or stressful events such as natural disasters, illness or death of a relative, miscarriage and relationship conflict is correlated with a variety of objective and subjective health markers.Methods/DesignThis study is embedded within the longitudinal Healthy Aging of Women’s study which has collected data from midlife and older Australian women at 5 yearly intervals since 2001, and uses the Allostastic model of women’s health by Groër and colleagues in 2010. The current study expands the focus of the HOW study and will assess the impact of life stressors on quality of life and clinical biomarkers in midlife and older Australian women to explain the impact of chronic psychological stress in women.DiscussionThe proposed study hypothesizes that women are at increased risk of exposure to multiple or repeated stressors, some being unique to women, and the frequency and chronicity of stressors increases women’s risk of adverse health outcomes. This study aims to further our understanding of the relationships between stressful life experiences, perceived quality of life, stress biomarkers, chronic illness, and health status in women.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1−/−) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1−/−and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1−/− mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 ± 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 ± 0.12 ng/ml and 2.91 ± 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1−/− HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1−/− HFD-fed mice, but not in NOD/SCID mice. In Rag1−/− HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of long-term Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1−/− mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia.

No effect of administration of unacylated ghrelin on subcutaneous PC3 xenograft growth in a Rag1-/- mouse model of metabolic dysfunction

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1−/− mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 μg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1−/− mice on a high-fat diet, but did not significantly improve other metabolic parameters. We hypothesise that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome. Conflict of interest The authors declare no conflict of interest.

Abstract 3491: A role for the long non-coding RNAGHRLOSin cancer

Long non-coding RNA (lncRNA) genes are abundant in the human genome, and many are now recognized as oncogenes and/or tumor suppressors. We previously characterized the structure of GHRLOS, an antisense gene on the opposite strand of the multifunctional ghrelin gene (GHRL), however, its expression and function in disease has not been described. To this end, using The Cancer Genome Atlas (TCGA) data set, we revealed that GHRLOS is differentially expressed in a number of cancers. In particular, expression was elevated in endometrial cancer (1.2 - fold; P = 7.1 x 10 -3 Welch9s two-sample t-test; n = 24 vs. n = 175) and prostate cancer (1.2-fold; P = 3.7 x 10 -6 ; n = 52 vs. n = 498) compared to normal tissues. Using qRT-PCR (and commercial cDNA panels) we confirmed that GHRLOS expression was upregulated in endometrial cancer (1.96-fold, P = 0.005 Welch’s two-sample t-test, n = 5 vs. n = 17) and prostate cancer (2.46-fold, P = 0.0045 Welch’s two-sample t-test, n = 5 vs. n = 21) compared to normal tissues. Initial studies, using siRNA designed to silence endogenous GHRLOS expression, significantly reduced cell migration in the PC3 prostate cancer cell line (0.47-fold change, P = 0.042 Kruskal-Wallis test, n = 2) conversely, preliminary data using forced GHRLOS overexpression increased migration and proliferation. Taken together, we show that the long non-coding RNA GHRLOS is differentially expressed in tumor tissue and regulates cell migration and proliferation; possibly by modulating alternative splicing of the overlapping, multifunctional ghrelin gene locus. Targeting GHRLOS could provide a valuable and novel way to target the ghrelin axis in disease. Ongoing studies aim to validate in vitro functional results in complementary mouse xenograft models and identify genes and pathways regulated by this novel lncRNA. Citation Format: Esha T. Shah, Penny Jeffery, Patrick Thomas, Inge Seim, Lisa Chopin. A role for the long non-coding RNA GHRLOS in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2017-3491