HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

Abstract

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.

Expression of the Reverse Transcriptase Domain of Telomerase Reverse Transcriptase Induces Lytic Cellular Response in DNA-Immunized Mice and Limits Tumorigenic and Metastatic Potential of Murine Adenocarcinoma 4T1 Cells

“…Derivatives of murine mammary gland adenocarcinoma cells expressing firefly luciferase 4T1luc2 (“Bioware Ultra Cell Line 4T1luc2,” Caliper, Hopkinton, MA, USA; ) were generated as described previously [ 42 ]. In brief, coding sequence for rtTERT was recloned from pET15rtTERT into lentiviral vector pRRLSIN.cPPT.PGK (Addgene plasmid #12252; a gift from D. Trono) under the control of the human phosphoglycerate kinase (PGK) promoter, generating lentiviral vector pLVrtTERT ( Supplementary Figure S1 ).…”

“…Resulting 4T1luc2 derivatives were cultured in the full RPMI-1640 medium with 10% FBS and 100 mg/mL penicillin/streptomycin mix at 37 °C with 5% CO 2 and split every 2–3 days. Doubling time of derivative clones was estimated as described previously [ 42 ]. Presence of inserts of rtTERT DNA was confirmed by PCR and sequencing.…”

“…The capacity of 4T1luc2 derivative clones expressing rtTERT to form tumors and metastases was tested by their ectopic implantation into 8-weeks-old female BALB/c mice done as was described previously [ 42 ]. Briefly, suspensions of parental or daughter rtTERT expressing 4T1luc2 clones in the doses of 2.5 × 10 3 , 5 × 10 3 , and 1 × 10 4 cells in 50 μL of RPMI-1640 were injected into mice to the right and to the left of the base of the tail ( n = 4 per dose per cell line).…”

Reverse transcriptase inhibition potentiates target therapy in BRAF-mutant melanomas: effects on cell proliferation, apoptosis, DNA-damage, ROS induction and mitochondrial membrane depolarization

“…Finally, we reasoned that the thread linking between the DNA damage, the alteration of mitochondrial membrane potential and induction of apoptosis may be the accumulation of oxidative stress in melanoma cells [42,43]. Towards this goal, we took advantage of FACS analyses using dihydroethidium (DHE), a reagent that measures the reactive oxidative species allowing to distinguish cellular subpopulations into ROS positive (+) or negative (−) cells.…”

Reverse transcriptase inhibition potentiates target therapy in BRAF-mutant melanomas: effects on cell proliferation, apoptosis, DNA-damage, ROS induction and mitochondrial membrane depolarization

“…Finally, we reasoned that the thread linking between the DNA damage, the alteration of mitochondrial membrane potential and induction of apoptosis may be the accumulation of oxidative stress in melanoma cells (33)(34)(35). Towards this goal, we took advantage of FACS analyses using dihydroethidium (DHE), a reagent that measures the reactive oxidative species allowing to distinguish cellular subpopulations into ROS positive (+) or negative (-) cells.…”