Introduction

Hepatocellular Carcinoma (HCC) is the most common form of primary liver cancer accounting for 90% of cases and is the 6^th leading cause of death in the U.S. Texas has the highest HCC age-adjusted incidence rate at 45% higher than the nation’s average for 2001 to 2015 presenting a significant challenge. HCC has etiological concerns with nonalcoholic fatty (steatohepatitis) liver disease (NAFLD) and NAFLD is often associated with metabolic comorbidities such as obesity-related Type-2 diabetes (commonly treated with metformin initially). Metformin has demonstrated preventative measures against HCC and previous studies report having a high BMI in adolescence or adulthood increases ones’ risk for HCC, yet the connection between metabolic syndrome and HCC epidemiology remains to be understood. Furthermore, accumulating evidence suggests a link between affective/mood disorders (e.g., depression, bipolar disorder, anxiety, and chronic stress) and pathophysiological responses associated with metabolic-related diseases, but mechanistic knowledge gaps remain in linking psychological stress to HCC and its related health outcomes.

Chronic stress exposure modifies signaling pathways in the CNS via the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal axis resulting in systemic pathophysiological alterations capable of increasing risk for cancer morbidity/mortality. Previously, a clinical study determined that HCC patient quality of life scores significantly declined following diagnosis and were more prone to describe worsening mental health. Although evidence indicates that psychosocial stressors stimulate immunological responses in cancer development, progression, and mortality, there are limited studies investigating the role of chronic psychological stress and its reduction in HCC patient health outcome. Studies have reported similarities between mitochondrial dysfunction and pathophysiological characteristics of chronic stress. Cortisol may trigger genomic instability in mitochondria as a determinant of HCC tumor pathobiology in concert with cortisol-mediated effects on immune cell mitochondrial function. FKBP5 and TSC22D3 regulate stress responses and chronic glucocorticoid treatment is believed to establish a chronic stress phenotype, enabling further investigation into its effects on mitochondrial and immunological functions in HCC.

Methods

Treatment. Huh7.5.1 HCC cells were treated with control (DMEM, 10% FBS, 1% Pen/Strep), vehicle (0.04% ethanol/EtOH), or hydrocortisone concentrations of 0.1 µM (0.004% EtOH), 0.5 µM (0.02% EtOH), and 1 µM (0.04% EtOH). Treatments were administered for up to 10 days, after which cells were collected 24 hours following the final treatment.

RT qPCR. RNA was extracted with Trizol Reagent and cDNA was generated via the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor. Duplex qPCR reactions were performed with TaqMan^Ⓡ Gene Expression Assays targeting ACTB (housekeeping), and FKBP5 or TSC22D3 to determine relative gene expression levels.

Results

The huh7.5.1 cell line can be seeded at extremely low densities (2.2% confluency) and be maintained until 90-100% confluent. Preliminary results show TSC22D3 gene expression continuously decreases under hydrocortisone treatment for up to four days and increases after five and six days of treatment.

Conclusion

Preliminary results suggest increase in TSC22D3 expression levels upon acute hydrocortisone treatment (five-six days) may indicate initial development of a GC-induced phenotype. Future research will assess mitochondrial and immunological function/activity to determine mechanisms contributing to progressive interactions within the tumor microenvironment in hopes of identifying preventative measures and potential therapeutic targets for improving patient care outcome for HCC in individuals at risk for mental health disorders.