Expression of HIV‑1 reverse transcriptase in tumor cells of epithelial origin modulates their mitochondrial respiration and motility

Background: HIV-1 infected people are characterized by high prevalence of cancers affecting epithelial cells despite successful antiretroviral treatment. We hypothesized that this is due to the direct carcinogenic properties of HIV-1 proteins, including reverse transcriptase (RT) [1]. The aim of this study was to dissect the mechanism(s) underlying RT carcinogenicity by evaluating the effect of HIV RT expression on the metabolic activity and motility of tumor cells of epithelial origin.

Materials and methods: RT_A expressing subclones of 4T1luc2 cells were described previously [2]. Ca Ski cells (CRL-1550) were transduced with lentiviral particles expressing RT of HIV clade A FSU_A strain RT_A described earlier [2]. Resulting cells were cloned by single cell culturing, subclones were assessed for RT expression by PCR and Western blotting. ROS production was registered with 2′,7′-dichlorodihydrofluoresceine diacetate. Expression of E-cadherin, N-cadherin, Vimentin, Twist, Snail, and tubulin A and G was assessed by RT Q-PCR with SYBR Green on a RotorGene6000 cycler (Qiagen). Wound healing assay (WHA) was performed using Cytation 5 (BioTek) for 18 h with 1 h interval. Mitochondrial respiration and glycolysis were assessed using Seahorse technology (Agilent). Data were analyzed using nonparametrical statistics (GraphPad Prism 6), p < 0.05 was assigned significant.

Results: In mammary gland adenocarcinoma 4T1 cells, RT expression led to increased production of ROS, enhanced cell motility, and overexpression of mRNA of EMT factors including Twist, dependent on the levels of RT expression. In metabolic assays, RT-expressing 4T1 cells demonstrated enhanced basal respiration and spare respiratory capacity with no significant changes in glycolysis. Implanted into BALB/C mice, RT-expressing 4T1 cells caused faster tumor growth and higher metastasic activity than parental 4T1 cells (p < 0.05). Tumor size and metastatic activity were proportional to RT expression. In cervical cancer Ca Ski cells, stable expression of RT led to suppressed basal respiration and decreased respiratory capacity, increased glycolysis, and at the same time, decreased the expression of mRNA of Twist, Nrf2, and tubulins A and G, and reduced directional cell motility. At the same time, RT-expressing Ca Ski demonstrated increased capacity to heal large wounds and loss of contact inhibition at the late stages of WHA, both characteristics of respiration and motility consistent with the distruction of microtubuli.

Conclusions: Expression of HIV-1 RT causes changes in mitochondrial respiration. Change in respiration parameters varies depending on the type of expressing cells, with enhancement of respiration and cell motility in epithelial breast cancer, and suppression of respiratory capacity, basal cell respiration and cell motility in cervical cancer cells. Fine interplay exists between respiration, mitochondrial morphology, and microtubule and microfilament polymerization, reduced basal respiration arising as a symptom of cytoskeletal impairment [3]. Cumulatively, our data indicate that HIV-1 RT is heavily involved in this interplay. Differential outcome of this involvement may be due to differences in the functions of microtubuli and regulation of mitochondrial respiration in the breast cancer cells, which are metabolically very plastic [4], compared to HPV-infected cervical cancer cells with a typical (more rigid) cancer metabolic profile [5].

Acknowledgements: Russian Fund for Basic Research 19_04_01034; Russian Science Foundation grant 19-74-10086.

References

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