Abstract

Simple Summary

   FOXP3 is a critical transcription factor that works as a master
   regulator of the lymphoid lineage. The expression of FOXP3 was also
   observed in tumor cells; in cervical cancer, FOXP3 increases as the
   tumor progresses. However, the biological role of FOXP3 in cervical
   pathology is not well understood. In addition, FOXP3 has isoforms that
   could have different biological properties. In this work, the
   expression of FOXP3 and its isoforms were evaluated. It was found that
   the isoform FOXP3Δ2Δ7 is expressed in the cervical cancer-derived cell
   line SiHa. The transduction of this isoform in nontumorigenic
   keratinocytes induces proliferation, cell division, and migration.
   RNAseq analysis indicated that the FOXP3Δ2Δ7 isoform induces the
   expression of different protooncogenes and modulates essential pathways
   related to the immune response and the tumorigenic process.

Abstract

   Cervical cancer (CC) is the fourth most common type of cancer among
   women; the main predisposing factor is persistent infection by
   high-risk human papillomavirus (hr-HPV), mainly the 16 or 18 genotypes.
   Both hr-HPVs are known to manipulate the cellular machinery and the
   immune system to favor cell transformation. FOXP3, a critical
   transcription factor involved in the biology of regulatory T cells, has
   been detected as highly expressed in the tumor cells of CC patients.
   However, its biological role in CC, particularly in the keratinocytes,
   remained unclarified. Therefore, this work aimed to uncover the effect
   of FOXP3 on the biology of the tumoral cells. First, public databases
   were analyzed to identify the FOXP3 expression levels and the
   transcribed isoforms in CC and normal tissue samples. The study’s
   findings demonstrated an increased expression of FOXP3 in HPV16+ CC
   samples. Additionally, the FOXP3Δ2 variant was detected as the most
   frequent splicing isoform in tumoral cells, with a high differential
   expression level in metastatic samples. However, the analysis of FOXP3
   expression in different CC cell lines, HPV+ and HPV-, suggests no
   relationship between the presence of HPV and FOXP3 expression. Since
   the variant FOXP3Δ2Δ7 was found highly expressed in the HPV16+ SiHa
   cell line, a model with constitutive expression of FOXP3Δ2Δ7 was
   established to evaluate its role in proliferation, migration, and cell
   division. Finally, RNAseq was performed to identify differentially
   expressed genes and enriched pathways modulated by FOXP3Δ2Δ7. The
   exogenous expression of FOXP3Δ2Δ7 promotes cell division,
   proliferation, and migration. The transcriptomic analyses highlight the
   upregulation of multiple genes with protumor activities. Moreover,
   immunological and oncogenic pathways were detected as highly enriched.
   These data support the hypothesis that FOXP3Δ2Δ7 in epithelial cells
   induces cancer-related hallmarks and provides information about the
   molecular events triggered by this isoform, which could be important
   for developing CC.

   Keywords: FOXP3, FOXP3Δ2Δ7, cervical cancer, HPV, RNAseq, GSEA

1. Introduction

   Cervical cancer (CC) is one of the most preventable and treatable types
   of cancer due to the extended precancerous phase that lasts decades,
   yet it is still recognized as a health burden in low- and middle-income
   countries where it is ranked as the fourth most common type of cancer
   [[34]1,[35]2] and the second leading cause of cancer death, both in
   women and men [[36]3]. The most frequent histopathologic subtype of CC
   is squamous cell carcinoma (SCC), which accounts for nearly 75–85% of
   the cases, and it is derived from the transformation of squamous cells
   at the external portion of the cervix, termed ectocervix, and the
   adenocarcinoma (ADC), with approximately 10–25% of CC cases, which is a
   consequence of columnar epithelial cells transformation at the
   endocervix, the internal portion of the organ [[37]4,[38]5,[39]6];
   there is also a third subtype named as adenosquamous carcinoma (ADSC),
   which accounts for merely 2–3% and is originated by a merge of both
   squamous and glandular cells [[40]5].

   The main risk factor for CC is persistent infection by high-risk human
   papillomavirus (hr-HPV), such as the 16 and 18 genotypes, which both
   are associated with approximately 70% of cases worldwide [[41]7,[42]8];
   HPV16 is frequently associated with SCC, while HPV18 is associated with
   ADC [[43]9]. Hr-HPVs stimulate cell transformation over decades through
   the constant expression of their oncoproteins to activate immunological
   and carcinogenic pathways to favor tumorigenesis and modulate local
   immunity [[44]10,[45]11,[46]12,[47]13].

   FOXP3, forkhead box p3, is a member of the transcription factor family
   known as forkhead/winged helix; its locus is in the short arm of the X
   chromosome, which transcribes for an mRNA of 11 protein-coding exons.
   Interestingly, by alternative splicing, five isoforms could arise from
   the FOXP3 mRNA; these are FOXP3-FL, FOXP3Δ2, FOXP3Δ7, FOXP3Δ2Δ3, and
   FOXP3Δ2Δ7 [[48]14,[49]15]. FOXP3 is recognized as a master regulator
   responsible for immune tolerance through the modulation of genetic and
   functional programs in regulatory T cells (Treg) [[50]16]. The increase
   in Tregs in cancer is associated with a worse prognosis due to their
   immunosuppressive functions. The suppressive properties of Tregs rely
   on stable FOXP3 expression [[51]16,[52]17,[53]18]. The two major
   isoforms of FOXP3 reported in Tregs are FOXP3-FL and FOXP3Δ2, which are
   expressed in approximately equal amounts; the main functional
   difference between both isoforms is derived from the inability of
   FOXP3Δ2 to inhibit RORα and RORγt functions [[54]19,[55]20,[56]21]. A
   recently published study suggested that the expression of FOXP3 is
   closely related to the occurrence and growth of cervical cancer
   [[57]22]. The expression of FOXP3 correlates with the prognosis of
   cervical cancer, and it is significantly higher in cancer than in
   cervical intraepithelial neoplasia or chronic cervicitis [[58]22].
   However, it remains unanswered whether there is a correlation between
   Tregs FOXP3+ and hr-HPV infection [[59]23]. Nowadays, it is known that
   the FOXP3 expression is not exclusive of lymphoid lineage as it has
   been detected in epithelial cells from the breast, lung, prostate
   [[60]24], and retinal tissue [[61]25], as well as in cancer cells
   [[62]26,[63]27].

   The role of FOXP3 in tumor cells is, however, controversial. It has
   been observed in breast and prostate cancer cells that FOXP3 expression
   is associated with antitumoral roles [[64]28,[65]29,[66]30,[67]31].
   Furthermore, in ovarian cancer cells, inhibitory properties in
   proliferation, migration, and invasion were observed when FOXP3 was
   upregulated [[68]32]. Conversely, the FOXP3 increase has also been
   linked to protumor functions in pancreatic, colorectal, gastric,
   bladder, thyroid, cervical, and nonsmall cell lung cancer
   [[69]33,[70]34,[71]35,[72]36,[73]37,[74]38]. In CC, the FOXP3
   expression increased as the lesion progressed [[75]39]. In addition,
   the knockdown of FOXP3 diminishes the growth of the SiHa cells
   [[76]40]. However, whether the FOXP3 expression is regulated by HPV,
   which isoform is expressed in normal and tumoral keratinocytes, and the
   implications of FOXP3 expression in nontumorigenic epithelial cells are
   questions that remain unanswered. Therefore, this work aimed to
   evaluate the role of FOXP3 in the biology of keratinocytes and its
   contribution to the development of CC.

2. Materials and Methods

2.1. Cell Culture

   SiHa, CaSki, HeLa, SW756, C33A, HaCaT, and Lenti-X 293T cell lines were
   cultivated in Dulbecco’s Modified Eagle Medium (DMEM) with D-glucose
   (4.5 g/L) (No. Cat. 10-013-CV, Sigma-Aldrich, St. Louis, MI, USA),
   L-glutamine (584 mg/L), penicillin (100 U/mL), streptomycin (100
   μg/mL), sodium pyruvate (110 mg/L), and 10% fetal bovine serum (FBS).
   Cells were maintained at 37 °C and a 5% CO[2] atmosphere in an
   incubator (C170UL-120V-R, Binder, Tuttlingen, Germany).

2.2. FOXP3Δ2Δ7 Open Reading Frame Cloning

   The open reading frame (ORF) of FOXP3Δ2Δ7 was amplified through
   conventional PCR with DreamTaq Green PCR Master Mix (Cat. No. K1081,
   Thermo Scientific, Waltham, MA, USA) according to the manufacturers’
   instructions; cDNA from SiHa cells was used as a template. Primer
   sequences were designed using the Primer-BLAST tool from NCBI [[77]41]
   ([78]Table 1). The PCR product (1347 pb) was resolved by
   electrophoresis in 0.8% agarose gel, cut, and purified with Zymoclean
   Gel DNA Recovery Kit (Cat. No. D4001/D4002, ZYMO RESEARCH Corporation,
   Irvine, CA, USA) according to their instructions. The amplicon was
   ligated into the pGEM-T Easy vector (Cat. No. A137A, Promega, Madison,
   WI, USA) with T4 DNA Ligase (Cat. No. M180A, Promega, Madison, WI, USA)
   overnight at 4 °C. The ligation reaction was transformed into competent
   TOP10 bacteria by thermal shock and selected by the white/blue method.
   The constructed plasmid was named pGEM-FOXP3Δ2Δ7; from this, plasmid
   preparations were performed using the QIAprep Spin Miniprep Kit (Cat.
   No. 27106, QIAGEN Inc., Valencia, CA, USA).

Table 1.

   Primers sequences.
   Gene Forward Reverse Amplicon Size
   FOXP3 ^1 5′ ACA AGC CAG GCT GAT CCT T 3′ 5′ CAC ATC CAG GGC CTA TCA TC
   3′ 1347 bp
   FOXP3 ^2 5′ CAA GTT CCA CAA CAT GCG ACC 3′ 5′ GCT CTC CAC CCG CAC AAA
   3′ 208 bp
   SATB1 ^2 5′ CCT CAG CCA GAA CGT GAT GC 3′ 5′ GAC TCT GCT GGA GAG GCC A
   3′ 236 bp
   C1R ^2 5′ AAG ATT CCT CGG TGC TTG CC 3′ 5′ GTT GCT TTG CGC TTC GTG TT
   3′ 216 bp
   GLI2 ^2 5′ CAA CAA TGA CAG TGG CGT GG 3′ 5′ CTG CCA CTG AAG TTT TCC AGG
   3′ 297 bp
   LAMP3 ^2 5′ ACA TGC GGT GGT GAT GTT CC 3′ 5′ AGG CAG AGA CCA ACC ACG AT
   3′ 219 bp
   NSG1 ^2 5′ TTC CTC ACC TGC GTC GTC TT 3′ 5′ AAC TTG CCC ATC CCG CTA AG
   3′ 297 bp
   HSPB8 ^2 5′ GGT GGC ATT GTT TCT AAG A 3′ 5′ TAC TGG CAT CTC AGG TAC AG
   3′ 208 bp
   RPLP0 ^2 5′ CCT CAT ATC CGG GGG AAT GTG 3′ 5′ GCA GCA GCT GGC ACC TTA
   TTG 3′ 95 pb
   RPL32 ^2 5′ GCA TTG ACA ACA GGG TTC GTA G 3′ 5′ ATT TAA ACA GAA AAC GTG
   CAC A 3′ 320 pb
   [79]Open in a new tab

   ^1 Primer used for gene cloning. ^2 Primer used for quantitative PCR.

   Subsequently, the FOXP3Δ2Δ7 ORF was isolated from the pGEM-FOXP3Δ2Δ7
   vector by restriction with EcoRI (No. Cat. IVGN0116, Invitrogen,
   Waltham, MA, USA) and subcloned into the lentiviral vector pLVX-Puro
   (Cat. No. 632164, Clontech Laboratories Inc., Mountain View, CA, USA)
   that was previously linearized with EcoRI, and dephosphorylated with
   Antarctic Alkaline Phosphatase (Cat. No. IVGN2204, Invitrogen, Waltham,
   MA, USA). The resulting plasmid pLVX- FOXP3Δ2Δ7 was used to sequence
   the FOXP3Δ2Δ7 ORF using the Big Dye v3.1 Terminator Cycle Sequencing
   kit (Cat. No. 4337455, Applied Biosystems, Thermo Fisher Scientific
   Inc., Waltham, MA, USA), and the ABI PRISM 310 Genetic Analyzer
   (Applied Biosystems). The sequences were aligned using the UGENE
   software (v.1.21.1) [[80]42]. The NCBI sequences [81]NM_014009.3 and
   [82]NM_001114377.1 were used as references.