Role of Host Mirna Hsa-Mir-139-3P in HPV-16–Induced Carcinomas M.K

Published OnlineFirst January 31, 2017; DOI: 10.1158/1078-0432.CCR-16-2936

Biology of Human Tumors Clinical Cancer Research Role of Host miRNA Hsa-miR-139-3p in HPV-16–Induced Carcinomas M.K. Sannigrahi1, Rajni Sharma2, Varinder Singh1, Naresh K. Panda1, Vidya Rattan3, and Madhu Khullar2

Abstract

Purpose: Human papillomavirus 16 (HPV-16) is an important ation and cell migration, cell-cycle arrest at G2–M phase and risk factor in head and neck cancer (HNC). Studies suggest that increased cell death of HPV-16–positive cells. Analysis of The miRNAs play an important role in cancer; however, their role in Cancer Genome Atlas (TCGA) data showed decreased expression HPV-mediated oncogenesis remains largely unknown. We inves- of Hsa-miR-139-3p in HPV-16–positive HNC and cervical cancer tigated the role of miRNAs with HPV-16 as putative target in HPV- cases, and its higher expression correlated with better survival 16–mediated cancers. outcome in both cases. Increased DNA methylation of Hsa- Experimental Design: Using in silico tools, we identified miR-139-3p harboring gene PDE2A at its promoter/CpG islands miRNAs with putative binding sequences on HPV-16 miRNAs. was observed in HPV-16–positive tissues and cell lines, which Hsa-miR-139-3p was identified as best candidate miRNA by further correlated with Hsa-miR-139-3p expression, suggesting luciferase reporter assay and was found to be significantly its role in regulating Hsa-miR-139-3p expression. Furthermore, downregulated in HPV-16–positive tissues and cell lines. Over- we observed an increased sensitization of Hsa-miR-139-3p overexpression/inhibition studies were performed to determine the expressed HPV-16–positive cells to chemotherapeutic drugs (cis- role of miRNA in regulating oncogenic pathways. platin and 5-fluorouracil). Results: Hsa-miR-139-3p was found to target high-risk HPV-16 Conclusions: HPV-16–mediated downregulation of Hsa- oncogenic proteins and revive major tumor suppressor proteins miR-139-3p may promote oncogenesis in HNC and cervical (p53, p21, and p16). This resulted in inhibition of cell prolifer- cancer. Clin Cancer Res; 23(14); 3884–95. 2017 AACR.

Introduction infection by several types of viruses like human immunodeﬁcien- cy virus (6), primate foamy virus type 1 (7), and vesicular Human papillomaviruses (HPV) represents an important eti- stomatitis virus (8). It is suggested that viruses may alter ological agent for cervical cancer and head and neck cancer. The miRNA expression of infected cells, resulting in deregulation of virus expresses two main oncogenic proteins, E6 and E7 that target host defense pathways and helping invading viruses to establish host tumor suppressor proteins p53 and pRB, respectively (1, 2). an environment favorable for persistence of viral infection (9, 10). Besides this, HPVs also induce various genetic and epigenetic Vertebrates encode several hundred miRNA families with changes in host leading to carcinogenesis (3). distinct seed sequences and it has been suggested that two-third Virus–host interactions are critical in determining host suscep- of these vertebrate miRNAs may have at least one hit in a virus tibility/resistance to viral infections (4, 5). miRNA form an genome of approximately 16 kb by random chance alone (11). important part of mammalian innate antiviral immunity Furthermore, knocking down the miRNA-processing enzymes response and were found to act as restriction factors to limit such as Drosha and Dicer which reduces the processing of mature mammalian miRNAs has been shown to lead to more

1 robust viral replication indicating their role in host–viral inter- Department of Otolaryngology, Post Graduate Institute of Medical Education – and Research (PGIMER), Chandigarh, India. 2Department of Experimental Med- actions (12 14). Furthermore, infecting viruses have been icine and Biotechnology, PGIMER, Chandigarh, India. 3Unit of Oral Health shown to alter miRNA expression of infected cells, resulting in Sciences, PGIMER, Chandigarh, India. de-regulation of host defense pathways and helping invading Note: Supplementary data for this article are available at Clinical Cancer viruses to establish an environment favorable for the persistence Research Online (http://clincancerres.aacrjournals.org/). of viral infection (15). Several recent studies have reported Prior presentations: The work has been presented in various national and differential expression of microRNAs in HPV infected cell lines international conferences. Part of the work is published as an abstract in and HPV-positive tumor tissues (16–18) and it has been sug- conference proceedings, including AACR Special Conference on Noncoding gested that aberrant miRNA expression may be regulated by RNAs and Cancer: Mechanisms to Medicines; December 4–7, 2015, Boston, MA, a epigenetic mechanisms like DNA methylation in HPV-asso- conference on new ideas in cancer-challenging dogmas, and 35th Annual ciated cancers (19). We hypothesized that HPV-16–induced Convention of Indian Association for Cancer Research. differential expression of host miRNAs may help its survival Corresponding Author: Madhu Khullar, Post Graduate Institute of Medical and activation of HPV-16 associated oncogenic pathways. Our Education and Research, Chandigarh 160012, India. Phone: 172-274-2842; Fax: results show that downregulation of Hsa-miR-139-3p, targeting 172-274-4401; E-mail: [email protected] HPV-16 viral transcription, activates HPV-16 oncogenic E6 doi: 10.1158/1078-0432.CCR-16-2936 and E7 proteins, and may be involved in HPV-16–induced 2017 American Association for Cancer Research. carcinogenesis.

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obtained from RegRNA program and Micro-Inspector program Translational Relevance having putative target in HPV-16 mRNAs, those miRNAs 0 HPV-16 is one of the emerging and major risk factors in (100 miRNAs) having 2–8 seed sequence at the 3 -end and head and neck cancer and cervical cancer. We have shown a with binding free energies under the threshold of 20.0 kcal/ novel viral–host interaction mechanism through cross-talk mol were initially identified and screened for their HPV-16 between HPV-16 and Hsa-miR-139-3p. Putative miRNAs tar- mRNA specificity by RNA-Hybrid program and for putative geting HPV-16 mRNA were identified and validated in clinical targets on host mRNAs by TargetScan software version 6.2. samples, TCGA database, and in vitro studies in head and neck 10 best miRNAs were shortlisted on the basis of high number cancer and cervical cancer cases. We observed that of target sites in HPV-16 mRNAs and least putative target sites in * Hsa-miR-139-3p had four putative targets in E1 region of human mRNAs (Supplementary Information S2). Primary cul- HPV-16 early mRNA. ture of keratinocytes were used as control (Supplementary * Hsa-miR-139-3p overexpression resulted in decreased Information S3) and differential expression of these miRNAs expression of HPV-16 mRNAs/proteins (E6/E7). were further analyzed under different conditions (Supplemen- * Hsa-miR-139-3p overexpression also resulted in decreased tary Information S4) to identify best candidate miRNAs having p16, revival of p53 and p21 expression. putative target on HPV-16 mRNAs. * Hsa-miR-139-3p overexpression decreased cell IHC staining, Western blot analysis, and antibodies proliferation, cell migration, and G2–M arrest, leading to cell death. Tissue samples were examined by IHC/Western blotting (WB) fi * Decreased expression of Hsa-miR-139-3p is due to for HPV-16-speci c pathway proteins using Anti-p16 (sc-9968; promoter methylation of its gene. Santa Cruz Biotechnology), anti-p53 (sc-6243; Santa Cruz Bio- * Hsa-miR-139-3p sensitizes HPV-16–positive cells to technology), anti-pRb (sc-73598; Santa Cruz Biotechnology), chemotherapy. anti-E6 (sc-460; Santa Cruz Biotechnology), anti-p21 (sc-6246; We propose that HPV-16–mediated promoter methylation Santa Cruz Biotechnology), and anti-GAPDH (sc-25778; Santa fi of Hsa-miR-139-3p gene may result in downregulation of Cruz Biotechnology) antibodies. Quanti cation was done using Hsa-miR-139-3p in HPV-16–infected cells, leading to upregu- ImageJ software (Supplementary Information S5). lation of pro-oncogenic pathways and HPV-16-induced car- Transfection with Hsa-miR-139-3p mimic/inhibitor/scrambled cinogenesis. Thus, identification of such antiviral miRNAs control that are epigenetically silenced by HPV-16 will help in devel- Hsa-miR-139-3p mimic (referred as mimic, catalog no. 4464084; oping epigenetic therapy against HPV-induced cancer. Further, Thermo Fisher Scientific) and Hsa-miR-139-3p inhibitor (referred using such miRNAs in combination with common che- as inhibitor, catalog no. 4464066; Thermo Fisher Scientific) were motherapies can lead to de-escalation of dose of chemo-drugs used to accordingly increase and decrease endogenous Hsa-miR- and will be beneficial to patients. 139-3p levels, respectably by transfection using Lipofectamine 2000 (catalog no. 11668; Invitrogen). Cells were treated with 100 pmol/mL of mimic/inhibitor. Scrambled miRNA (catalog no. 4464058; Thermo Fisher Scientific) was used as negative control. Materials and Methods Dual luciferase assay Patient specimens and cell cultures Luciferase assay was performed in HPV-negative UPCI:SCC 116 HNC tissue samples were collected from patients with sus- cells using noncommercial dual-luciferase enzyme assay system pected malignancy attending minor OT, Department of Otolar- (26). Firefly luciferase (FL) plasmid pMiR-HPV16-E7 (with region yngology, PGIMER (Chandigarh, India), after obtaining informed 562 to 658 encoding E7-CDS, Addgene #53696) and pMiR- consent from individual patients. Control tissue group consisted HPV16-E1 (with region 865 to 2813 encoding E1-CDS, Addgene of patients undergoing surgery for non-neoplastic diseases of #53699; ref. 27), and Renilla pRL-SV40P (Addgene #27163; head and neck in Department of Oral Health Sciences, PGIMER ref. 28). Renilla luciferase pRL-SV40P plasmid was used as an (Chandigarh, India). internal control. Five-hundred nanograms of each plasmid and Three HPV-negative cell-lines (UPCI:SCC-116, HaCat, and 100 pmol of Hsa-miR-139-3p scrambled/mimic/inhibitor was – HEK-293) and three HPV-16 positive cell-lines (UPCI:SCC- transfected into cells. 090, SiHa, and CaSki) were cultured (20). Cell proliferation, migration, and apoptosis analysis HPV-16 detection in tissue samples Cell proliferation was measured using Click-iT Plus EdU cell HPV-16wasdetectedinDNAsamplesusing:MY09-MY11pri- proliferation assay (catalog no. C10632; Life Technologies; þ þ mers, Gp5 /Gp6 primers, and HPV-16-E6-specific primers. HPV-16 ref. 29). Cell death and cell cycle was determined by Dead Cell viral load and relative expression of HPV-16-E7 mRNA was measured Apoptosis Kit with Annexin V FITC and PI (catalog no. V13242; by using qRT-PCR (ref. 21; Supplementary Information S1). Life Technologies). Cells were examined in a FACSCalibur flow cytometer (BD Biosciences). Cell migration was analyzed by fi In silico identi cation of miRNAs targeting HPV-16 in vitro scratch assay (30). The width of scratch wounds was mea- The seed sequences of the miRNAs were screened for ones that sured using ImageJ software (Supplementary Information S6). could potentially directly target HPV-16 mRNAs using MicroIn- spector (22), RegRNA program (23), and RNAHybrid program TCGA cases (24). Putative miRNA targets on host mRNAs were predicted using miRNA profile and clinical details of HNC and CC patients TargetScan software version 6.2 (25). Out of the >600 miRNAs of TCGA were obtained from Firehose Broad GDAC (http://gdac.

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broadinstitute.org/). A total of 255 cervical squamous cell carci- overexpression/inhibition. In brief, cell lines with 4,000 cells/well noma cases were included for analysis. A total of 279 HNC were seeded in 96-well plates were transfected with 50 pmol Hsa- samples already analyzed for HPV (31, 32) were included in miR-139-3p mimic/inhibitor. After 24 hours, cells were treated study, which were further grouped into HPV-negative HNC with 5, 10, and 20 mmol/L of chemotherapeutic drugs for 48 hours (n ¼ 243) and HPV-16–positive (n ¼ 29). Other HPV type and cell viability was measured (Supplementary Information S9). positive cases (n ¼ 7) were excluded. Methylation data of HNC (n ¼ 261) and CC (n ¼ 177) cases were obtained from MethHC Statistical analysis database (ref. 33; Supplementary Information S7). Statistical tests like nonparametric Mann–Whitney test, two- tailed t test, ANOVA, and survival analysis done using GraphPad DNA methylation and acetylation analysis Prism 5 (Graph Pad Software). A P value of <0.05 was denoted a 0 Cells were treated with 5-Aza-2 -deoxycytidine (10 mmol/L) statistically significant difference. and Trichostatin A (100 ng/mL) for 4 days, and media containing 5-aza-dC/TSA was changed every 24 hours. For methylation Ethical approval analysis, Genomic DNA (200 ng) converted with the EZ-DNA Approved by Institutional Ethics Committee (IEC), Post Grad- Methylation Kit (Zymo Research) and amplified using methyla- uate Institute of Medical Education and Research (PGIMER), tion-specific Hsa-miR-139-3p primers and TaKaRa EpiTaq HS Chandigarh, India. (catalog no. R100A; Clontech Laboratories). For acetylation analysis, chromatin immunoprecipitation (ChIP) assays were performed using ChIP-IT Express Enzymatic Kit (catalog no. Results 53009; Active-Motif; Supplementary Information S8). A total of 110 suspected cases of HNC were analyzed for HPV/ HPV-16 infection using PCR (Supplementary Information S1) In vitro chemosensitivity assay and were grouped into HPV-negative HNC (n ¼ 40), Control Cells were treated with different doses of cisplatin (CDDP) and (HPV-negative, n ¼ 40), and HPV-16–positive HNC (n ¼ 30). 5-fluorouracil (5-FU) combination for 48 hours and cell viability HPV-16 DNA–positive patients were further grouped into HPV-16 was determined using MTT assay to calculate IC50/LD50. Finally, E7 mRNA positive (n ¼ 20) and HPV-16 E7 mRNA negative (n ¼ their effect was observed in combination with Hsa-miR-139-3p 10). The demographic profile of the patients is shown in Table 1.

Table 1. Demographic characteristics of patients Groups Hsa-miR-139-3p expression HPV-16 E7 mRNA-positive group Hsa-miR-139-3p up Hsa-miR-139-3p down Patient characteristics Control HPV-16–negative HNC HPV-16–positive HNC E7 mRNA-positive E7 mRNA-negative P value Total HNC patients 40 40 30 20 10 Age <60 y 32 (80) 33 (82.5) 25 (83.3) 17 (85) 8 (80) 0.551 60 y 8 (20) 7 (17.5) 5 (16.7) 3 (15) 2 (20) Gender Male 30 (75) 32 (80) 25 (83.3) 18 (90) 7 (70) 0.191 Female 10 (25) 8 (20) 5 (16.7) 2 (10) 3 (30) Subsite Oral cavity 15 (37.5) 12 (40) 7 (35) 5 (50) Oropharynx NIL 14 (35) 13 (43) 8 (40) 5 (50) Hypopharynx 5 (12.5) 2 (7) 2 (10) 0 Larynx 5 (12.5) 3 (10) 3 (15) 0 Overall stage Stage 0 þ 1 NIL 0 0 0 0 1 Stage 2þ3þ4 40 (100) 30 (100) 20 (100) 10 (100) Smoking Never smokers 10 (25) 5 (12.5) 5 (16.7) 4 (20) 1 (10) 1 Light smokers 12 (30) 8 (20) 10 (33.3) 6 (30) 4 (40) 0.600 Heavy smokers 18 (45) 27 (67.5) 15 (50) 10 (50) 5 (50) 1 Alcohol No 20 (50) 15 (37.5) 17 (56.7) 10 (50) 7 (70) 0.440 Yes 10 (25) 25 (62.5) 13 (43.3) 10 (50) 3 (30) Chewing Non-chewers 32 (80) 28 (70) 22 (73.3) 15 (75) 7 (70) 1 Chewers 8 (20) 12 (30) 8 (26.7) 5 (25) 3 (30) Viral load High NIL NIL 14 (46.7) 12 (60) 2 (20) 0.044a Low 16 (53.3) 8 (40) 8 (80) Integration Integrated NIL NIL 15 (50) 13 (65) 2 (20) 0.002a Mixed 8 (26.7) 6 (30) 2 (20) 0.040a Episomal 7 (23.3) 1 (5) 6 (60) 1 p16 staining Positive NIL NIL 14 (46.7) 14 (70) 0 0.000a Negative 16 (54.3) 6 (30) 10 (100) aStatistically signiﬁcant, P < 0.05.

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In silico selection of host miRNAs with HPV-16 mRNAs as in HPV-16–positive UPCI:SCC-090 (14-fold, P < 0.01), SiHa putative targets (122-fold, P < 0.01), and CaSki cell lines (19-fold, P < 0.01), and miRNAs with binding sites in HPV-16 mRNAs were screened upregulated in HPV-negative UPCI:SCC 116 cells (4.2-fold) when based on their binding free energies (< 20.0 kcal/mol), "seed compared with primary culture (Fig. 1A; Supplementary Infor- region" (six to eight nucleotides at 50 end) and Hydrogen bond mation S3). Furthermore, its expression was not affected by index. They were shortlisted on the basis of high number of target external factors including cell growth media (Supplementary sites in HPV-16 mRNAs and least putative target sites in human Information S4). mRNAs (Supplementary Information S2). In silico analysis revealed that Hsa-miR-139-3p had four putative targets in E1 region of HPV-16 mRNA and no targets In vitro identification of miRNA-targeting HPV-16 sites in E7 region (Fig. 1B; Supplementary Information S4). Hsa-miR-139-3p identified as candidate miRNA. Of the 10 short- These observations were further validated by Luciferase Assay listed miRNAs, Hsa-miR-139-3p was significantly downregulated in which Hsa-miR-139-3p mimic transfected HPV-negative

Figure 1. Hsa-miR-139-3p expression in HPV-16–positive and HPV-negative cell lines. A, Relative expression of Hsa-miR-139-3p in HPV-16-positive (UPCI:SCC 090, SiHa, CaSki) cell lines, primary keratinocytes, and HPV-negative (UPCI:SCC 116) cells. B, Putative targets of Hsa-miR-139-3p in HPV-16 E1 mRNA region. C, Relative FL repressive effect was analyzed using four FL plasmid constructs: pMiR as vector control, pMiR-E1 containing the putative sites, and pMiR-E7 as external vector control with viral DNA and no putative sites of Hsa-miR-139-3p. HPV-negative UPCI:SCC 116 cells were cotransfected with 1 mg of pMiR-plasmids and Renilla pRL-SV40P plasmid, and 100 pmol/L scrambled (S), Hsa-miR-139-3p mimic (M), and Inhibitor (I). Renilla luciferase was used as an internal control to normalize FL activity expression. Results were compared with scrambled miRNA transfected cells for statistical analyses. Plasmid containing the E1 region of primary HPV-16 mRNA showed signiﬁcant decreased FL activity. , P < 0.05; , P < 0.01.

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A B HPV Negative HPV Positive 1.8 UPCI:SCC 116 UPCI:SCC 090

HPV 16 E6 1.2 p16 0.6 p21

0.0 p53

mRNAs (E6/E7:18S) GAPDH

Relative expression of HPV-16 Relative expression Mimic_E7 Mimic_E6 + – –––+ Inhibitor_E7 Inhibitor_E6 Scrambled Scrambled_E7 Scrambled_E6 100 pmol/L Mimic – ++––– 100 pmol/L Hsa-miR-139-3p mimic/inhibitor 200 pmol/L Mimic ––+ ––+

CDE

HaCat SCC-116SiHa SCC-090CaSki HaCat SCC-116SiHa SCC-090CaSki HaCat SCC-116SiHa SCC-090CaSki HPV 16 E6 HPV 16 E6 HPV 16 E6

p53 p53 p53

p16 p16 p16

p21 p21 p21 GAPDH GAPDH Scrambled miRNA transfection Scrambled GAPDH Hsa-miR-139-3p mimic transfection Hsa-miR-139-3p inhibitor transfection

Figure 2. Hsa-miR-139-3p overexpression/inhibition leads to signiﬁcant changes in expression of targeted genes in HPV-positive cells. A, Relative expression of HPV16 mRNA (E6 and E7 primers) in UPCI:SCC 090 cells transfected with 100 pmol Hsa-miR-139-3p mimic (Mimic_E7 and E6) compared with scrambled control (Scrambled_E7 and E6) and 100 pmol Hsa-miR-139-3p Inhibitor (Inhibitor_E7 and E6). , P < 0.01. B, Western blot analysis of HPV-16 E6 proteins and HPV-targeted pathway proteins: p53, p16, and p21 in HPV-negative UPCI:SCC 116 and HPV-positive UPCI:SCC 090 cells transfected with 100 and 200 pmol Hsa-139-3p Mimic. C, Western blot analysis in HPV-negative cells (HaCat, UPCI:SCC 116) and HPV-16–positive cells (SiHa, UPCI:SCC 116, CaSki) transfected with 100 pmol of scrambled miRNA, (D) Hsa-miR-139-3p mimic, and (E) Hsa-miR-139-3p inhibitor.

UPCI:SCC-116 cells showed 30% to 35% decrease in FL activity Hsa-miR-139-3p overexpression/inhibition modulated host in case of pMiR-E1 plasmid (P < 0.01) and 15% to 18% oncogenic processes decrease in pMiR-E1þE7 (P < 0.01, 1:1) compared with control Hsa-miR-139-3p overexpression decreased cell proliferation (Scrambled miRNA) and Hsa-miR-139-3p inhibitor–trans- rate in HPV-positive cells (49%, CaSki and 38%, SiHa) and fected cells (Fig. 1C). its inhibition increased cell proliferation rate (14%, CaSki; 79%, SiHa). However, no significant change in proliferation was Hsa-miR-139-3p overexpression/inhibition modulated HPV observed in HPV-negative cells (Fig. 3A). pathway genes Hsa-miR-139-3p overexpression resulted in decreased cell via- Hsa-miR-139-3p overexpression in HPV-16–positive UPCI: bility of HPV-16–positive cells (20%, SiHa; 27%, UPCI:SCC-090) SCC-090 cells decreased HPV-16 mRNA (E6 and E7) levels (6.5- and inhibition increased cell viability (35%, SiHa; 6%, UPCI: to 6.9-fold, P < 0.01; Fig. 2A). Moreover, Hsa-miR-139-3p SCC-090; Fig. 3B). overexpression in HPV-16–positive UPCI:SCC-090 cells also Hsa-miR-139-3p overexpression increased accumulation of affected expression of HPV pathway proteins in a dose-depen- cell at G2–M phase in HPV-16–positive cells (105%, UPCI: dent manner (Fig. 2B and Supplementary Information S5). Hsa- SCC-090 and 201%, SiHa) as compared with HPV-negative cells miR-139-3p overexpression decreased expression of HPV-16 E6 (7.2%, UPCI:SCC 116) and inhibitor transfected cells (13% to (1.8-fold, P < 0.05), p16 (5.5-fold, P < 0.05), and increased p53 31%; Fig. 3C). (20-fold, P < 0.05) and p21 expression (10.6 fold, P < 0.05) in Hsa-miR-139-3p overexpression resulted in significantly HPV-16–positive cells (SiHa, UPCI:SCC-090, and CaSki) as increased cell death in case of HPV-16–positive cells (84.6%, compared with control (scrambled miRNA-transfected HaCat UPCI:SCC-090; 55%, SiHa) when compared with HPV-negative and UPCI:SCC-116 cells; Fig. 2C–E). Hsa-miR-139-3p inhibitor cells (5.8%, UPCI:SCC-116; 7.3%, HaCat) and Hsa-miR-139-3p showed no significant changes. inhibition (Fig. 3D).

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Figure 3. Overexpression and inhibition of Hsa-miR-139-3p in HPV-16–positive and HPV-negative cells. HPV-negative cells (UPCI:SCC 116 and HaCat) and HPV-16–positive cells (UPCI:SCC 090, SiHa, and CaSki) transfected with 100 pmol scrambled/mimic/inhibitor. A, Graph shows EdU stained cells after FACS analysis. B, Graphs show the average viability of cells in MTT cell viability assay relative to the appropriate scrambled control (taken as 100%). C, Graphs show FACS analysis of pI stained transfected with 100 pmol of Hsa-miR-139-3p mimic compared with scrambled transfected cells. D, Graph showing average percentage of dying cells. E, Graph shows percentage of space between two monolayes after scratch assay at different time intervals. HPV-16 positive. , P < 0.05.

Hsa-miR-139-3p overexpression signiﬁcantly decreased cell tumor-suppressors (p53, p21), and resulting in decreased cell migration as longer time interval for gap closure was observed proliferation, G2–M arrest, and cell death in HPV-16–infected in HPV-16–positive cells as compared either scrambled miRNA or cells. miRNA inhibitor or Hsa-miR-139-3p overexpressed HPV-negative cells (Fig. 3E). Ex vivo analysis of Hsa-miR-139-3p expression These results suggest Hsa-miR-139-3p acts as antiviral miRNA, Hsa-miR-139-3p expression downregulated in HPV-positive tissue restricting HPV-16 oncoprotein expression (E6, E7), recovers samples. Hsa-miR-139-3p expression was also found to be

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Figure 4. Relative expression of Hsa-miR-139-3p in HPV-16–positive HNSCC tissue samples. A, Relative expression of Hsa-miR-139-3p from TCGA data in HPV-negative HNC (n ¼ 243), HPV-16–positive HNC (n ¼ 29), and HPV-positive CC (n ¼ 254). B, Relative expression of Hsa-miR-139-3p in HPV-16 DNA-positive HNSCC (n ¼ 30), HPV-negative HNSCC (n ¼ 40), and Oral Health controls (n ¼ 40) biopsy samples. C, Relative expression of Hsa-miR-139-3p in HPV-16–positive E7 mRNA-positive samples (n ¼ 20). D, Corelation of Hsa- miR-139-3p expression, viral load, and E7 mRNA expression in HPV-16–positive samples (n ¼ 20). , P < 0.05; , P < 0.01; , P < 0.001.

significantly downregulated in HPV-16–positive HNC (n ¼ 27) TCGA survival data correlated decreased Hsa-miR-139-3p expres- and HPV-positive cervical cancer (n ¼ 254) with respect to HPV- sion with active HPV infection. Cervical cancer patients are 99% negative samples (n ¼ 238) from TCGA (Fig. 4A). HPV-positive with poor survival rate (34). However, we observed In our HNC cohort (n ¼ 110), Hsa-miR-139-3p expression was cervical cancer cases with increased Hsa-miR-139-3p expression downregulated 12-fold in HPV-16 DNA positive HNC (n ¼ 30) (high, n ¼ 82, relative-expression >5.93) had significantly better with respect to HPV-negative HNC tissue samples (n ¼ 40, P < survival (HR 0.7; 95% CI, 0.4–1.2; P < 0.05) than those having 0.001) and 21-fold with respect to control (HPV-negative non- decreased Hsa-miR-139-3p expression (low, n ¼ 136, relative- cancerous) tissue samples (n ¼ 40, P < 0.001; Fig. 4B). Out of expression <5.93; Fig. 5A). 30 HPV 16 DNA-positive samples, 20 (66.7%) showed HPV-16 HPV-16–positive HNC have significantly better survival E7-mRNA amplification suggesting an active infection. Hsa-miR- (HR 0.4; 95% CI, 0.2–0.8; P < 0.01) than HPV-negative 139-3p expression was also significantly downregulated (P < HNC (P < 0.01; Fig. 5B). However, HPV16-positive HNC 0.05) in E7 mRNA–positive HNC tissues (n ¼ 20) as compared with increased Hsa-miR-139-3p expression (high, n ¼ 9, with E7 mRNA negative HNC tissues (Fig. 4C). We also observed relative-expression >15.04) had better survival (HR 0.8; that E7 mRNA expression is negatively correlated with Hsa-miR- 95% CI, 0.07–8.3; P > 0.05) than those having decreased 139-3p expression [r ¼0.07, P ¼ 0.4; 95% confidence interval Hsa-miR-139-3p expression (low, n ¼ 12, relative-expression (CI), 0.5–0.4; Fig. 4D). Furthermore, HPV-16 E7–positive HNC <15.04; Fig. 5C). tissues (n ¼ 20) had significantly higher viral load and integrated form of viral DNA. The viral load ranged from <1 to 537.6 103 Regulation of expression of Hsa-miR-139-3p in copies per 103 cells. Hsa-miR-139-3p expression had significant HPV-positive HNC negative co-relation with viral load, that is higher viral load In silico analysis revealed Hsa-miR-139-3p lies within the had lower Hsa-miR-139-3p expression and vice-versa (r ¼ intronic region of PDE2A (Chromosome 11; 72615130- 0.42, P ¼ 0.03; 95% CI, 0.73–0.04; Fig. 4D). Hsa-miR-139- 72615063). Further from TCGA data, PDE2A expression 3p expression was also decreased in p16 IHC positive (n ¼ 14), showed significant co-relation with Hsa-miR-139-3p expres- p53-negative (n ¼ 10), and/or pRB-negative (n ¼ 13) biopsy sion in cervical cancer (n ¼ 153) and HNC (n ¼ 249), suggest- samples (Supplementary Information S4). ing both may be coordinately regulated (Fig. 6A). MethHC data These result suggest decreased Hsa-miR-139-3p expression co- show a negative correlation between PDE2A expression and related with active HPV infection. promoter/CpG methylation in HNC and cervical cancer

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A Survival curve: Survival proportions in CC B Survival curve : with high and low Hsa-miR-139-3p expression HPV-16+ve HNC vs HPV -ve HNC 150 100

75 100 Hsa-miR-139 High HPV16+ve HNC 50 50 HPV-ve HNC

Percent survival Percent 25 Hsa-miR-139 Low survival Percent

0 0 0 2,000 4,000 6,000 8,000 0 1,000 2,000 3,000 4,000 5,000 Days Time (days)

Survival curve :Survival proportions in HNC C with high and low Hsa-miR-139-3p expression 110 Low_HPV-16 100 High_HPV-16 90

80 70 Percent survival Percent 60

50 0 1,000 2,000 3,000 Days

Figure 5. Survival analysis in HNC and cervical cancer tissue samples from TCGA data. Kaplan–Meier survival curves of (A) HPV-positive cervical cancer patients with high Hsa-miR-139-3p expression (n ¼ 82)comparedwithlowHsa-miR-139-3pexpression(n ¼ 136). (B)HPV-16–positiveHNCpatients(n ¼ 21) compared with HPV-negative HNC patients (n ¼ 192). C, Low Hsa-miR-139-3p expressed HPV16–positiveHNCpatients(n ¼ 12)comparedwithHigh Hsa-miR-139-3p expressed HPV16–positive HNC patients (n ¼ 9). , P < 0.05; , P < 0.01.

(Supplementary Information S7). Comparing the methylation tissues and controls (HPV negative noncancerous; 2/26; levels from human pan-cancer methylation database, MethHC Fig. 6E). across gene regions around promoter and CpG islands showed Analysis of HPV-16 integration at promoter site of PDE2A significantly increased methylation in HPV-16–positive HNC did not reveal any integration of HPV-16 genome with PDE2A (n ¼ 29) and cervical cancer (n ¼ 175) with respect to HPV- (Supplementary Information S8). Further role of histone acet- negative (n ¼ 220) cases (Fig. 6B). ylation on Hsa-miR-139-3p expression was measured by TSA Further, 5-Aza-dC treatment of HPV-16–positive and HPV- treatment of HPV-16–positive cell lines (Fig. 6C) and by ChIP negative cell-lines was carried out to examine if it had effect in cell lines (Fig. 6E). However, no significant change in Hsa- on Hsa-miR-139-3p expression. 5-Aza-dC treated HPV-16– miR-139-3p expression was observed (Supplementary Infor- positive UPCI:SCC-090 cells showed significantly increased mation S8). Hsa-miR-139-3p expression (Fig. 6C; Supplementary Informa- Thus, our results suggest that promoter/CpG methylation reg- tion S8). ulates Hsa-miR-139-3p expression in HPV-16–positive epithelial Promoter/CpG methylation of PDE2A (Chromosome 11; cell lines and HNSCC biopsy samples. 72676856-72677047) was analyzed using methylation sensitive PCR (MSP; Supplementary Information S8). HPV-16– Hsa-miR-139-3p overexpression lowered positive cells (UPCI:SCC-090, CaSki, and SiHa) showed chemotherapeutic dosage significantly increased methylation as compared with Systemic treatment of HNC involves combination of cis- HPV-negative (HEK-293, HaCat, and UPCI:SCC 116) cell platin (CDDP) and 5-fluorouracil (5-FU) (35). Initially, lines, which was validated by sequencing (Fig. 6D). MSP LD50/IC50 values of CDDPþ5-FU (1:1) was determined in analysis of HNC tissue samples showed that 36% (9/25) of HPV-positive and HPV-negative cell lines. HPV-16–positive HPV-16–positive tissue samples showed promoter methyl- cell lines (UPCI:SCC-090, 31.34 mmol/L and SiHa, 26.65 ation as compared with 7.6% (2/26) HPV-negative HNC mmol/L) were observed to be more resistant to treatment as

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ABCorrelation in Head and Neck Cancer 4,000 0.4 3,000 0.3

2,000 0.2 1,000 P = 0.000*** 0.1 0 PDE2A Expression 0 50 100 150 200 0.0 Hsa-miR-139-3p Expression Tumor methylation Tumor at PDE2A promoter –0.1 Correlation in Cervical Cancer –0.2 1,000 800 600 400 P = 0.000*** CC_CpG 200 CC_Promoter 0 HPV-16+ve_CpG PDE2A Expression HPV-ve_HNC CpG 0 10 20 30 40 HPV-ve_HNC PromoterTCGA Tissue samples Hsa-miR-139-3p Expression HPV16+ve_HNC Promoter CDE

6 8 1.0

4 6

0.5 2 4 (miR-139-3p: U6) (miR-139-3p: (miR-139-3p: U6) (miR-139-3p: Relative expression 0 2 Relative expression 0.0 Intensity of methylation 0

AZA_116TSA_116 AZA_090TSA_090 INPUT HaCat_H3 CaSki_H3HaCat_H4 CaSki_H4 DMSO_SCC 116 DMSO_SCC090 SCC116_H3SCC090_H3 SCC116_H4SCC090_H4 HPV Negative HPV Positive Cell lines Cell lines Cell lines

Figure 6. Methylation and ChIP assays in cell lines and biopsy samples. A, Comparison of PDE2A expression and Hsa-miR-139-3p expression by linear regression analysis in HNC (n ¼ 264) and cervical cancer (n ¼ 153) cases from MethHC. B, Methylation levels of Hsa-miR-139-3p harboring gene PDE2A at promoter and CpG island region in HPV-16–positive (n ¼ 29), HPV-negative (n ¼ 236) HNC, and cervical cancer (n ¼ 172) cases. C, Relative expression of Hsa-miR-139-3p in cells treated with 5-aza-dC concentrations (10 mmol/L) and TSA (100 ng/mL) for 4 days. D, HPV-16–positive cell lines (UPCI:SCC-090, SiHa, and CaSki) and HPV-negative cell lines (HEK, HaCat, and UPCI:SCC-116) ampliﬁcation band intensities using methylated and unmethylated-speciﬁc primers were analyzed using ImageJ software. E, Relative expression of Hsa-miR-139-3p in HPV-16–positive (UPCI:SCC 090, CaSki) and HPV-negative (HaCat, UPCI: SCC 116) cell lines in ChIP. , P < 0.05; , P < 0.01; , P < 0.001.

compared with HPV-negative cell lines (UPCI:SCC-116, 8.626 Discussion mmol/L and HaCat, 8.894 mmol/L; Fig. 7A; Supplementary Information S9). Recent reports suggest that miRNAs play an important role in Further, on Hsa-miR-139-3p overexpression followed by host–viral interactions. In this study, we have addressed how HPV treatment of chemotherapeutic drugs (5 to 20 mmol/L) resulted may modulate host antiviral miRNAs to their own benefit. We fi in a 20% to 50% decrease in LD50 values (Fig. 7B–E) in HPV- identi ed Hsa-miR-139-3p, a host miRNA having putative target positive cells (SiHa, 11.16 mmol/L and UPCI:SCC-090, 11.06 on HPV-16 mRNA. Our results suggest that HPV-16 downregu- mmol/L) cells as compared with scrambled miRNA-transfected lates Hsa-miR-139-3p expression to create a favorable environ- HPV-16–positive cells (SiHa, 18.61 mmol/L and UPCI:SCC- ment for viral replication and oncogenesis. 090, 16.63 mmol/L) and Hsa-miR-139-3p overexpressed Although several miRNAs showed binding affinity for HPV-16 HPV-16–negative cells (HaCat, 17.08 mmol/L and UPCI:SCC- mRNA, only Hsa-miR-139-3p expression was significantly 116, 14.60 mmol/L), suggesting Hsa-miR-139-3p overexpres- decreased in HPV-16–positive HNC tissues and cell lines. This sion sensitizes cells to a CDDPþ5-FU treatment in HPV-16– is further supported by TCGA data, where Hsa-miR-139-3p was positive cell lines. found significantly downregulated in HPV-positive HNC and

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Host miRNAs Targeting HPV-16 HNSCC

Figure 7.

Dose–response curve analysis of inhibition of cell growth by the drug treatment correlated to drug concentrations and corresponding IC50/LD50 values. A, Cells were treated for 48 hours with various concentrations of combination of CDDP and 5-FU from 1 to 100 mmol/L (1, 2, 4, 8, 10, 20, 40, 80, and 100) to determine the IC50 or LD50 value for the combination treatment. B–E, Cells were transfected with 50 pmol Hsa-miR-139-3p mimic/inhibitor. After 24 hours, cells were treated with 5, 10, and 20 mmol/L concentration of cisplatin and 5-FU combination for 48 hours. cervical cancer cases. It is also reported to be dysregulated in Hsa-miR-139-3p in regulating HPV-16 replication during the cervical cancer (36), colon cancer (37), bladder cancer (38), and initiation of infection. Further, this region (between 1703 and leukemia (39). 2044) is present in most fusion transcripts including early gene ^ Hsa-miR-139-3p was found to target E1 region (between transcript from episomal HPV (E7-E1 E4) and several late ^ 1703 and 2044) of HPV-16 by luciferase reporter assay. Studies gene transcripts from integrated HPV (such as E7-E1 cellular ^ showed that E1 expression is critical for and limiting in the RNA, E7-E1 E4-cellular RNA, etc.) in HPV-16–infected tissues initial ampliﬁcation of the HPV-16 genome (40) and also for (42, 43). Thus, in those cases where HPV-16 has integrated into maintenance replication during keratinocyte differentiation genome, Hsa-miR-139-3p may also target late HPV-16 mRNAs (41). In accordance with these studies, a negative correlation whichinturnisknowntoregulatestheexpressionofother was observed between HPV-16 viral load and Hsa-miR-139-3p oncogenic proteins like E6 and E7 and thereby regulate onco- expression in HNC tissue samples, suggesting potential role of genesis. In accordance to these studies, we observed a decreased

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Sannigrahi et al.

relative E7 mRNA expression co-related with high Hsa-miR- Because Hsa-miR-139-3p was found to promote apoptosis of 139-3p expression in biopsy samples and vice-versa. To further HPV positive cells, we investigated if it could potentiate response examine its role, Hsa-miR-139-3p was overexpressed in HPV- to chemotherapy as microRNAs have been reported to be effective 16–positive cell lines and this resulted in suppression of E6 as adjunct therapy to chemo-/radiotherapy-resistant cells (49). and E7 which induced revival of tumor suppressors: p53 and We found that HPV-16–positive cells had higher LD50/IC50 p21, indicating its role in restricting HPV-16 expression. Sim- values, probably due to degradation/inhibition of p53/p21 by ilar results were observed in in vitro and in vivo studies where HPV oncogenes as chemotherapy drugs, CDDP and 5-FU are siRNAs used to directly silence E6/E7 expression (42–44), known to act through p53-mediated apoptotic pathways (50, suggesting Hsa-miR-139-3p regulates E6/E7 expression. More- 51). However, Hsa-miR-139-3p overexpression in HPV-16–pos- over, Hsa-miR-139-3p overexpression also decreased cell itive cells resulted in decreased LD50/IC50 values by 20% to 50%, growth/proliferation and increased cell death. This further indicating that Hsa-miR-139-3p was sensitizing these cells to validated antiviral role of Hsa-miR-139-3p as revival of p53/ chemotherapy. Thus, our results suggest that Hsa-miR-139-3p p21 pathway in viral-infected cells is known to mediate could be a potential adjunct therapy in chemotherapeutic treat- growth arrest, apoptosis, and necrosis (45, 46). Thus, Hsa- ment of HPV-16–infected patients. miR-139-3p acts as antiviral miRNA by limiting expression of In summary, our work on Hsa-miR-139-3p adds to the growing HPV-16 oncogenes. evidence of association between microRNAs and HPV in cancers. To investigate whether HPV-16 is directly modulating Hsa- Thus, we propose a model of HPV infection where-in down- miR-139-3p, an exvivoanalysis was done in HPV-16–positive regulation of Hsa-miR-139-3p by promoter methylation in HPV- HNC tissues samples which showed decreased Hsa-miR-139-3p 16–infected cells results in upregulation of pro-oncogenic path- expression correlated with increased HPV-16 viral load and ways leading to HPV-16–induced carcinogenesis. However, active viral infection (HPV-16-E7 mRNA expression), suggest- because HPV-16 are known to multiply in basal epithelium layer ing HPV-16 may be downregulating Hsa-miR-139-3p expres- and cell line model with high expression of HPV-16 oncogenes sion. To further understand the mechanisms behind down- (E6 and E7) and not expressing HPV-16 E1 may not reflect a regulation of Hsa-miR-139-3p, epigenetic mechanisms like normal HPV infection cycle during epidermal differentiation, histone acetylation and CpG/promoter methylation were hence studies on transgenic mouse model and 3D cell culture examined. In human genome, pre-miR-139 (that encodes models for defining its prognostic implications are warranted. mature miR-139-3p) is located on the human chromosome 11q13.4 region NC_000011.10 (72615063...72615130, com- Disclosure of Potential Conflicts of Interest plement) and this miRNA is encoded within the second intron No potential conflicts of interest were disclosed. of phosphodiesterase 2A (PDE2A) gene. Hsa-miR-139-3p expression correlated with expression of its harboring gene Authors' Contributions PDE2A in cervical cancer and HNC samples. While examining Conception and design: M.K. Sannigrahi, R. Sharma, N.K. Panda, V. Rattan, methylation status of PDE2A promoter/CpG islands, we M. Khullar observed a significantly increased methylation level at its pro- Development of methodology: M.K. Sannigrahi, R. Sharma, M. Khullar moter/CpG regions in HNC tissues by MSP. Increased tumor Acquisition of data (provided animals, acquired and managed patients, methylation levels were also observed in HPV-positive HNC provided facilities, etc.): M.K. Sannigrahi, V. Singh, N.K. Panda, V. Rattan Analysis and interpretation of data (e.g., statistical analysis, biostatistics, and CC cases from MethHC database indicating promoter/CpG computational analysis): M.K. Sannigrahi, R. Sharma methylation decreases Hsa-miR-139-3p expression. As HPV-16 Writing, review, and/or revision of the manuscript: M.K. Sannigrahi, is known to induce DNA methylation pathway to activate the R. Sharma, M. Khullar development of cancer (47, 48), it is evident that downregula- Administrative, technical, or material support (i.e., reporting or organizing tion of Hsa-miR-139-3p that targets HPV-mRNAs by promoter data, constructing databases): M.K. Sannigrahi, M. Khullar methylation may be a survival mechanism adopted by HPV-16 Study supervision: R. Sharma, N.K. Panda, V. Rattan, M. Khullar to create a more favorable environment for its replication. However, studies on host cellular proteins targeted by Hsa- Acknowledgments This study was carried out at the Genetics of Complex Disorder laboratory, miR-139-3p and other host miRNAs during such host–viral PGIMER, Chandigarh, India. UPCI:SCC-116 and UPCI:SCC-090 cell lines were interaction that may also play important role in these survival kind gifts from Dr. S. Gollin (University of Pittsburgh, Pittsburgh, PA). The mechanisms adopted by HPV-16 leading to oncogenesis are authors are grateful to all individuals who participated in this study and the also warranted. laboratory staff members for their skillful technical help. M.K. Sannigrahi Survival analysis using TCGA data showed HPV-16–positive thankfully acknowledges research fellowship from Indian Council of Medical patients with increased Hsa-miR-139-3p expression has better Research, New Delhi, India. < The costs of publication of this article were defrayed in part by the payment of survival (HR 1) than those with decreased Hsa-miR-139-3p advertisement – page charges. This article must therefore be hereby marked in expression in HPV-16 positive patients HNC and cervical accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cancer patients suggesting high Hsa-miR-139-3p expression is restricting HPV-16 expression which is in-turn limiting its Received November 24, 2016; revised January 10, 2017; accepted January 11, oncogenesis. 2017; published OnlineFirst January 31, 2017.

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Role of Host miRNA Hsa-miR-139-3p in HPV-16−Induced Carcinomas

M.K. Sannigrahi, Rajni Sharma, Varinder Singh, et al.

Clin Cancer Res 2017;23:3884-3895. Published OnlineFirst January 31, 2017.

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