HIV-1 Suppresses Notch-1 Expression in HPV-16+ CaSki Cells for Cancer Progression

Serena Judith DSouza (  dsouzaserena17@rediffmail.com ) National AIDS Research Institute Arati Mane National AIDS Research Institute Linata Patil National AIDS Research Institute Aazam Shaikh National Centre For Cell Science Madhuri Thakar National AIDS Research Institute Vandana Saxena National AIDS Research Institute Leila Fotooh Abadi National AIDS Research Institute Sheela Godbole National AIDS Research Institute Smita Kulkarni National AIDS Research Institute Raman Gangakhedkar Division of Epidemiology and communicable diseases, ICMR Padma Shastry National Centre For Cell Science Samiran Panda National AIDS Research Institute


Background
There is an increased risk of co-infections with human papilloma virus (HPV) with persistence of many carcinogenic subtypes in Human Immunode ciency Virus (HIV-1)-positive women [1]. They are more prone to invasive cervical cancers too [2,3]. Epidemiological studies from a cohort of HIV-1 positive women established an incidence of 35.3 % carcinogenic human papillomavirus (HPV) genotypes.
Oncogenic HPV-16 also escapes immune surveillance in HIV-1 positive women during combined antiretroviral therapy (cART) [4]. HPV vaccines have not yet been examined for the recurrence of cervical intraepithelial lesions in HIV-1 positive women [5].
HPV lifecycle orchestrates growth and differentiation events through HPV transforming genes-E6/E7 facilitating tumorigenesis [6]. During HIV-1 infection, the accessory protein Tat and HPV E6/E7 proteins exploit Notch signaling to exacerbate HPV-associated disease pathogenesis [7,8]. Transmembrane Notch-1 protein is essential for proliferation, differentiation and apoptosis at all stages of life. Notch-1 activation, through ligand binding from neighboring cell encounter, induces proteolysis and release of Notch-1 intracellular domain (NICD) into the nucleus, the main gene processing centre [8]. Concomitantly, Numb, an evolutionary conserved protein enhances Notch-1 signaling by repressing NICD ubiquitination [9]. NICD also binds with DNAbinding transcription factor protein, CSL to initiate Notch-1 downstream targets, Hes-1 and Hey-1 gene transcription associated with carcinogenesis and progression of cervical carcinoma [10]. Alternative spliced Numb iso variants, short (Numb S) and long (Numb L) also in uence Hes-1 expression [10].
Notch-1, Numb and EGFR have similar roles in cell fate speci cations including their involvement in cancers [11]. In-vitro studies in breast cancer cell lines demonstrated a positive link between Notch-1 and EGFR when transfected with HIV-1 [12]. Notch-1 regulates growth and differentiation through cyclin D, CDK2 and p21 in cell lines like T-cell acute lymphoblastic leukemia [13]. Cervical cancer cells simultaneously utilize Notch-1 [14] as well as hyper-express CXCR4, a co-receptor of HIV-1. The cross interference of intricate pathways converge together viz CXCR4/Notch-1, Wnt/Notch-1, Notch-1/Snail/Numb, Notch-1/EGFR etc are implicated in cancer progression, survival and chemotaxis via intracellular signaling /downstream mediators [15,16].
Recurrent, invasive cervical carcinomas due to HIV-1 infection and interference with cell cycle marker expression, prompted us to examine the effects of HIV-1 Tat and full HIV-1 genome on HPV-ve C33A cells and HPV-16 + CaSki cells using transient transfection. We demonstrate here for the rst time that both HIV-1 Tat and HIV-1 suppresses Notch-1 expression in CaSki (HPV-16 + ) cells through signi cant Hes-1induction and increased CDK2 expression for G 0 -G 1 accumulation at the expense of S phase. HIV-1 Tat ampli es EGFR and p21 expression though Cyclin D is depleted, whereas HIV-1 transfection decreases EGFR and p21 expression concomitant with over active Cyclin D. Elevated Cyclin D preserves proliferation for unbridled mitosis, G 2 -M arrest, genomic instability and cancer progression [17,18]. Taken together, HIV-1Tat shows a positive cross talk between Notch-1and EGFR which favors an aggressive phenotype whilst HIV-1 has a reverse link between Notch-1 and EGFR. (Fig. 5)

Cell lines
Adherent cervical tumor derived cell lines HPV-Negative C33A cells (HPV -ve for HPV DNA and RNA having cervical cancer phenotype)and HPV-16 + CaSki cells(with inherent HPV-16 + and HPV-18 + sequences and endogenous Notch-1), were used for transfection.

Cell culture
CaSki and C33A cell lines were procured from National Centre for Cell Sciences (NCCS), Pune. CaSki cell line was propagated in RPMI-1640 with sodium bicarbonate without L-Glutamine supplemented with 10 % fetal bovine serum and antibiotic, anti-mycotic solution. C33A cells were grown DMEM medium consisting of L-Glutamine, Sodium pyruvate, Glucose and Sodium bicarbonate.

Plasmids
The pLEGFPN1-(RV-Tat 86) plasmid was a gift from Dr. Francesca Peruzzi, Associate Professor, LSU Health Sciences, New Orleans LA. The pNL 4.3, a full length replication competent, infectious HIV-1 subtype B, was obtained through NIH AIDS Reagent Program.

Cell proliferation
Brie y, CaSki and C33A cells (10 4 /well) were seeded in 96micro well culture plates and maintained at 37°C in a CO 2 incubator. Cells were exposed to serial dilutions of plasmid DNA/DAPT and incubated overnight for 24 hrs. MTT solution (5 mg/mL) was added to the plate and then incubated for 4 hrs.
Supernatant was replaced with DMSO, and absorbance was measured at 550/630 nm. The viability was calculated in percentage with reference to control sets.

Recombinant cloning
Plasmids were transformed using DH-5α through heat shock (42-45°C) and rapid chilling on ice. After heat shock treatment, the plasmid DH-5α mixture was added to pre-warmed SOC or LB without antibiotics. LB agar plate containing ampicillin were used to grow the transformed bacteria, at 32°C. Colonies were further inoculated in 15 mL LB mixed with 65 % glycerol for stock preparation and plasmid ampli cation.

Plasmid Puri cation
Brie y, bacterial cells were pelleted, resuspended in 250 µL of Solution I ( as per the manufacturer's protocol-Gene Mark plasmid miniprep puri cation kit). With subsequent addition of 250 µL Solution II, and following 4-5 times mixing, Solution III (250 µL) was added. The spin columns containing lysate/s were centrifuged, rinsed and purged of puri ed DNA with preheated elution buffer. The DNA was quanti ed and stored at -20°C for transfection.

Transient Transfection
Transfections were carried according to the manufacturer's instructions (jetPEI® DNA transfection reagent, USA). Brie y, CaSki and C33A cells were seeded in T-25 asks (1 X 10 6 ) with 5 ml medium containing FCS and antibiotics. Quanti ed DNA-Six hundred ng/mL DNA was used for transfecting one million cells [19]. The media was replenished the next day and cell lines maintained overnight in a CO 2 incubator. Cells were detached for further analysis.

Cell cycle analysis
Transfected cells were scraped gently, washed twice in cold PBS and processed using BD Cycletest™ Plus DNA Kit as per the manufacturer's protocol. Brie y, cells were pelleted, mixed with Solution A (trypsin buffer) for 10 min followed by incubation with Solution B (trypsin inhibitor and RNase buffer) for 10 min.
Finally the cells were stained with 200 µL of cold Solution C (PI stain solution) kept for 10 min in the dark at 4 ºC and analyzed for DNA content by BD FACSAria™ (BD, USA).

DNA isolation
DNA from transfected cells was isolated using the manufacturer's protocol (Vivantis Technologies, Malaysia). Brie y, the cell pellet (5 X 10 6 cells) was resuspended in PBS, treated with Proteinase K and lysis enhancer in Tris buffer. After incubation at 65 ºC for 10 min the cells were transferred to the column, followed by column washing with wash buffer. DNA was eluted with preheated elution buffer and quanti ed using spectrophotometer.

Polymerase Chain Reaction (PCR)
Notch-1 PCR was optimized with DNA isolated from human fetal buccal mucosal (FBM) cell line procured from ACTREC, Navi Mumbai, India. Twenty µL reaction mix (1.2 µL DNA template, primer mix and PCR dye master mix (5Xia)) was subjected to the following conditions ; 95 ºC for 4 min, 45 cycles at 95 ºC for 20 secs, 60 ºC for 20 secs, two extension steps − 72 ºC for 20 secs and 2 mins. Notch-1 PCR protocol was also used for CaSki cells.
Tat PCR was performed with DNA isolated from CaSki and C33A cells using the following conditions; 95 ºC for 9 mins followed by 34 cycles at 95 ºC for 1 min, 53 ºC for 2 mins, two extension steps at 72 ºC for 1 min and 7 mins respectively. PCR products were resolved on a 2.0 % agarose gel and visualized on a UV transilluminator after staining with ethidium bromide.

Western blot analysis
C33A and CaSki cells were harvested, washed, and lysed with RIPA buffer supplemented with phenylmethylsulfonyl uoride (PMSF). Protein extracts (30 µg) per sample were subjected to 8 % SDS-PAGE and transferred to polyvinylidene di uoride membranes (EMD Millipore, USA). Membranes were blocked with 5 % nonfat skim milk for 1 h at room temperature, probed with the appropriate primary antibodies (Notch-1, Hes-1, CDK2, EGFR and GAPDH a endogenous protein) at 1:1000 dilution, washed, and then incubated with the corresponding goat anti-rabbit and anti-mouse secondary antibody (1:5000 dilution). The chemiluminescent signal was detected using ECL system (Intron, Seongnam, Korea).
Semi-quantitative analyses of the intensities of the protein bands were analyzed by using ImageJ software (NIH).

RNA Isolation
RNA was isolated from transfected cell lines as per the manufacturer's protocol (GeneMark Biolab, Thailand). Brie y, transfected culture cells were lysed with lysis methanol cocktail in 70 % ethanol. The lysate was loaded on the spin column and washed. After DNAse treatment, the columns were washed again with wash buffer. Puri ed RNA (30-50 µL) was collected, quanti ed by a NanoDrop 2000c (Thermo Fisher Scienti c, USA) and processed for qPCR.

Discussion
Notch family genes, crucial to normal embryogenesis are also implicated in cancers [20]. In HIV-1 /HPV co-infected women with cervical carcinoma, extracellular HIV-1 Tat clinically correlates with an aggressive carcinoma phenotype [21]. In HPV associated cancers, Notch-1 inhibition induces malignant transformation [ 6,22]. In our study, we report for the rst time that HIV-1 Tat signi cantly elicited Notch-1 inhibition in HPV-16 + CaSki cells through activation of Notch-1 signaling pathway, characterized by downstream target genes-Hes-1 transcriptional activator, and Hey-1, transcriptional repressor. Hey-1, appears to be depleted in CaSki cells a possible characteristic of transformed cell lines. Based on our data, we propose that HIV-1 Tat plausibly binds to EGF repeats on the extracellular Notch-1domain [22], consequently suppressing Notch-1 expression. Hes-1 and Hey-1 genes are involved in adhesion, invasion, angiogenesis and proliferative events during carcinogenesis [23,24]. In T-cell leukemia, Hes-1 binds to the nuclear Cyclin D promoter to elicit Notch-1signaling. But, Hes-1 induction and Cyclin D repression are obligatory for sustenance and cancer progression in T-cell leukemia. Our observations in HIV-1 Tat transfected HPV-16 + CaSki cells, corroborate the above studies [23,25]. HIV-1 Tat signi cantly induces Hes-1 expression for Cyclin D shut down (*p ≤ 0.05),with suppression of EGFR. Now, EGFR, the transcriptional target of Notch-1 [26], cooperates with downstream targets Cyclin D and CDK2 for growth, proliferation and cell cycle progression [27]. Additionally, Cyclin D, a liated as downstream target of multiple oncogenic pathways, is also implicated in G 1 cell cycle progression. Cell cycle analysis demonstrate that HIV-1Tat signi cantly activates EGFR with consequent signi cant G 0 /G 1 phase cell accumulation (* p ≤ 0.05 ; supplementary Table 2) in HPV-16 + CaSki cells as compared with the HPV-, C33A cells. Recent evidences highlight that Cyclin D depletion disturbs oxidative balance in cancer cells. Subsequently, cancer cells succumb to high lethal oxidative stress inducing irreversible senescence [27]. Hence, HIV-1 Tat adjusts the tumor microenvironment to maintain the proliferative state to evade irreversible senescence through Hes-1 activation, EGFR and p21 ampli cation driving cell migration and invasion for an aggressive less therapeutic phenotype in HPV-16 + CaSki cells [28,29]. The above qRT-PCR data signi cantly collates with cell cycle G 2 -M phase population of HPV-16 + CaSki cells with HPV-C33A cells (* p ≤ 0.05).
Additive effects of HIV-1 Vpr and Notch-1/ CXCR4 [16] partner together to inhibit p21 and juxtapose aberrant mitosis, G 2 -M arrest for induction of replication restart [31] for metastasis and cancer progression.

Conclusion
Our results describe a negative link between Notch-1and EGFR during HIV-1 induced cancer progression. Tailor-made drug paradigms for clinical interventions may possibly inhibit an aggressive phenotype during cancer progression.

Consent for publication
No writing assistance was provided.

Availability of the data and materials
All data generated or analysed during this study are included in this published article and its supplementary information les. This information are available on request.

Competing interest
All authors have no con icting interests to declare.