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. 2019 Dec 2:2019:6016278.
doi: 10.1155/2019/6016278. eCollection 2019.

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

Ekaterina Bayurova  1   2 , Juris Jansons  3   4 , Dace Skrastina  3   4 , Olga Smirnova  5 , Dzeina Mezale  3 , Anastasia Kostyusheva  6 , Dmitry Kostyushev  6 , Stefan Petkov  7 , Philip Podschwadt  7 , Vladimir Valuev-Elliston  5 , Sviataslau Sasinovich  7 , Sergey Korolev  8 , Per Warholm  9 , Anastasia Latanova  1   5 , Elizaveta Starodubova  1   5 , Amir Tukhvatulin  1 , Oleg Latyshev  1 , Renat Selimov  10 , Pavel Metalnikov  10 , Alexander Komarov  10 , Olga Ivanova  5 , Tatiana Gorodnicheva  11 , Sergey Kochetkov  5 , Marina Gottikh  8 , Ilze Strumfa  3 , Alexander Ivanov  5 , Ilya Gordeychuk  1   2   12 , Maria Isaguliants  1   2   3   7
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HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist (V体育平台登录)

"V体育官网入口" Ekaterina Bayurova et al. Oxid Med Cell Longev. .

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 VSports手机版. 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. .

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Alignment of the amino acid sequences of the consensus HIV-1 reverse transcriptase and its drug resistance variants designed in the study. “RT_A,” consensus of amino acid sequences (n = 44) of HIV-1 subtype A FSU_A strain isolated between 1999 and 2012, with amino acids in variable positions chosen using OMES-based covariance network. “RT_An,” RT_A variant with introduced mutations of resistance to NRTI K65R and M184V; “RT_Ann,” RT_A variant with introduced mutations of resistance to NNRTI K103N and G190S; “K03455.1 HIV-1 subtype B (HXB2),” reference sequence of HIV-1 subtype B RT used for position numbering [63].
Figure 2
Figure 2
Expression of reverse transcriptase (RT) by 4T1luc2-derived cell lines carrying genomic inserts of sequences encoding variants of the consensus HIV-1 FSU_A RT. (a) Western blot analysis of lysates of derivative 4T1luc2 clones (Table 2) stained with rabbit polyclonal anti-RT antibodies [68] (I and II upper panels), and anti-actin monoclonal antibodies (I and II lower panels), recombinant RT_A as positive control (III). The parental 4T1luc2 cell line was used as a reference. Polyclonal anti-RT antibodies weakly bind to reverse transcriptase expressed by the lentivirus used to generate the parental 4T1luc2 cell line (II). Position of the weight mass marker is shown on the left. (b) Quantification of RT_A expression using ImageJ software. Signal in the lane corresponding to a given derivative clone was quantified using calibration curve built with the help of recombinant RT_A. Total amount of the expressed RT_A variant was divided by the number of cells used to make the lysate (see Materials and Methods for description). Data represent the results (mean ± SD) from three independent experiments.
Figure 3
Figure 3
Derivatives of 4T1luc2 cells expressing variants of consensus HIV-1 FSU_A reverse transcriptase (Table 2) exhibit increased levels of ROS production (a–c) and lipid peroxidation (d). ROS production was measured using fluorescent dye DCFH2-DA and normalized to the signal generated by DAPI staining. Total levels of ROS per cell on day 6 of cell culture; production of ROS is quenched by treatment with antioxidants tocopherol (1 μM) and NAC (5 mM); red asterisk indicates significant differences between 4T1luc2_RT-Ann-1.5 and other RT-expressing variants (p < 0.05) (a). Total levels of ROS production per cell on day 14 of cell culture relative to the levels exhibited by the parental 4T1luc2 cells (b). ROS levels on day 14 of cell culture normalized to the amount of RT_A variant (in fg) produced by one cell (c). Level of lipid peroxidation on day 14 of cell culture assessed as the concentration of MDA in the lysate of a single cell normalized to the amount of RT_A variant (in fg) produced by one cell (d). Values represent mean ± SD from two independent assays run in duplicates. ns: not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 (Kruskal-Wallis, followed by Mann-Whitney tests or ordinary one-way ANOVA followed by unpaired t-test).
Figure 4
Figure 4
Performance of the derivatives of 4T1luc2 cells expressing variants of consensus HIV-1 FSU_A reverse transcriptase (Table 2) in the wound healing assay. (a) The length of the gap (wound) in μm, at the time of the scratch and 14 and 18 hours later. (b) Distance covered by the cells during the first 14 hours after the scratch, calculated as a difference in the length of the gap at the time of the scratch and after 14 hours. (c) Distance covered by the cells in the interval between 14 and 18 hours after the scratch. (d) Speed of cell migration during the first 14 hours after the scratch, calculated as the covered distance divided by 14 h. (e) Speed of cell migration in the interval between 14 and 18 hours after the scratch. The length of the gap (distance covered by the cells) was calculated based on the light microscopy images using ImageJ software. All values are expressed as mean ± SD from five independent experiment runs. ns: not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
Figure 5
Figure 5
Relative expression levels of EMT markers in the derivatives of 4T1luc2 cells expressing variants of consensus HIV-1 FSU_A reverse transcriptase (Table 2) after 3 and 14 days in culture. Expression level was normalized to HPRT1 expression and calculated as fold change compared to the parental 4T1luc2 line. Results are presented as mean ± SD from two independent assays run in duplicates. ns: not significant; ∗∗∗∗p < 0.0001 by the ordinary two-way ANOVA.
Figure 6
Figure 6
Relative levels of expression of Twist by the derivatives of 4T1luc2 cells expressing variants of consensus HIV-1 FSU_A reverse transcriptase (Table 2) after 14 days in culture (a) and correlation of the relative expression of Twist with the levels of expression of RT_A variants (b). Expression level of Twist was normalized to the expression of HPRT1 and calculated as fold change compared to the levels exhibited by the parental 4T1luc2 cells. Values are presented as mean ± SD from the assay run in triplicates. The amount of RT_A variant produced per cell and expression of Twist at day 14 were correlated using Spearman ranking test (R = 0.9, p = 0.002); ∗∗p < 0.01.
Figure 7
Figure 7
Formation of solid tumors by the derivative clones of 4T1luc2 expressing variants of HIV-1 FSU_A reverse transcriptase (RT_A) after ectopic implantation into BALB/c mice. In vivo bioluminescent images showing growth of representative tumors formed by clones 4T1luc2_RT-1.3 (I), 4T1luc2_RT-5.3 (II), 4T1luc2_RT-20.1 (III), 4T1luc2_RT-An-1.4 (IV), 4T1luc2_RT-An-10.1 (V), 4T1luc2_RT-Ann-1.5 (VI), 4T1luc2_RT-Ann-10.2 (VII), and parental cells 4T1luc2 (VIII); red circles show the regions of interest (ROI) from which the total luminescent signal was collected (a). Growth of tumors assessed as the average total photon flux from the sites of injection encircled by ROI (b), the average percent change in the total photon flux compared to the photon flux from the site obtained on day 1 (c), the total photon flux from each of the injections sites on days 6 (d) and 18 (e) as percent of that on day 1, and palpable tumor size at the experimental endpoint (f). All group values represent the mean ± SD. After day 3, 4T1luc2_RT-1.3 and 4T1luc2_RT-5.3 derivative clones demonstrated significantly higher total photon flux, and 4T1luc2_RT-20.1 tended to have higher photon flux (p = 0.1) than the parental 4T1luc2 cells. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, and ∗∗∗∗p < 0.0001, Kruskal-Wallis, followed by Mann-Whitney tests.
Figure 8
Figure 8
Correlations (Spearman correlation test) between the tumor sizes represented by the total photon flux from tumor on day 18 and the levels of expression of RT_A variant per cell (R = 0.76, p < 0.05) (a); the levels of expression of Twist (normalized against the expression of HPRT1 and represented as fold increase compared to that of the parental 4T1luc2 cells) (R = 0.74, p < 0.05) (b); and palpable tumor volume on day 18 and change in the levels of expression of Twist from day 3 to day 14 in % (R = 0.96, p = 0.0001) (c).
Figure 9
Figure 9
Histochemical characterization of the solid tumors formed by the parental 4T1luc2 cells (a) and their derivatives expressing variants of HIV-1 FSU_A reverse transcriptase (RT_A) after ectopic implantation into BALB/c mice: 4T1luc2_RT-1.3 (b), 4T1luc2_RT-5.3 (c), 4T1luc2_RT-20.1 (d), 4T1luc2_RT-An-1.4 (e), 4T1luc2_RT-An-10.1 (f); 4T1luc2_RT-Ann-1.5 (g), 4T1luc2_RT-Ann-10.2 (h). H&E staining: magnification ×400.
Figure 10
Figure 10
Localization of luciferase-expressing cells in the organs of mice implanted with 4Tluc2 and derivative clones expressing variants of HIV-1 FSU_A reverse transcriptase (RT_A) after ectopic implantation into BALB/c mice as assessed by ex vivo bioluminescent imaging of the lungs (a), liver (b), and spleen (c). The values represent the mean total flux (p/s) ± SD (n = 3). ∗Significant difference from the value exhibited by the respective organ in 4Tluc2-implanted mice (p < 0.05; Mann-Whitney test).
Figure 11
Figure 11
Histochemical characterization of the formation of metastases in the livers of BALB/c mice ectopically implanted with 4Tluc2 and derivative clones expressing variants of HIV-1 FSU_A reverse transcriptase (RT_A). H&E staining of liver sections of mice injected with 4T1luc2 and its RT-expressing subclones (×400): 4T1luc2 (a), 4T1luc2_RT-1.3 (b), 4T1luc2_RT-5.3 (c), 4T1luc2_RT-20.1 (d), 4T1luc2_RT-An-10.1 (e), 4T1luc2_RT-Ann-10.2 (f). Mean number (g) and size (h) of metastases. Mean size of liver metastasis formed by all RT_A-expressing clones compared to the parental 4Tluc2 cells (i). Correlations between the mean numbers of metastases in a mouse liver assessed by histochemical staining and the levels of expression by the respective 4T1luc2 clone of Twist (day 14 of cell culture) (j) and of RT_A variant per cell (k). Ten high-power fields per mouse (n = 3) were screened to assess the number of metastasis; their size was evaluated using NIS-Elements software (Nikon, Tokyo, Japan), both values were represented as mean ± SD. Expression level of Twist was normalized to the expression level of HPRT1 and represented as fold change compared to the level in the parental 4T1luc2 cells. Comparisons were done using the Mann-Whitney test, and correlations, using the Spearman correlation test. ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, and ∗∗∗∗p < 0.0001.
Figure 12
Figure 12
BALB/c mice ectopically implanted with 4Tluc2 and derivative clones expressing HIV-1 FSU_A reverse transcriptase (RT_A) have grossly enlarged spleens (a) and a disproportion in the leukocyte subpopulations in the spleen (b). Spleen weight, in mg (a) and proportion of leukocytes in the spleen (b) of BALB/C implanted with given 4T1luc2 derivative clones (n = 3-9) was assessed at the experimental end-point, and represent mean ± SD. Data from naïve BALB/c mice (n = 3-5) is given for comparison. ∗p < 0.05; ∗∗p < 0.005 (two-way ANOVA test).
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