Abstract

Simple Summary

   Cervical cancer is one of the most lethal types of cancer in women from
   developing countries. These tumors are caused by long term infection
   with some human papillomavirus (HPV) types. Commonly, cervical cancer
   precursor lesions express high levels of matrix metalloproteinases.
   These enzymes break down specific components of the extracellular
   matrix affecting tissue structure and stiffness and cell motility.
   Matrix metalloproteinases and their natural inhibitors, such as
   Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK)
   protein, are important in normal tissue maintenance and remodeling and
   play a major role in the transformation process. Here, we showed that
   RECK over expression reduced the tumorigenic capacity of cervical
   cancer cells in vivo. In addition, tumors over expressing RECK
   presented altered inflammatory infiltrating cells when compared to
   controls. Our findings are useful to further understand the biology of
   cervical cancer and can help to determine if RECK may be a good
   therapeutic target for cervical cancer treatment in the future.

Abstract

   Human papillomavirus (HPV)-induced carcinogenesis comprises alterations
   in the expression and activity of matrix metalloproteinases (MMP) and
   their regulators. Reversion-inducing Cysteine-rich protein with Kazal
   motifs (RECK) inhibits the activation of specific metalloproteinases
   and its expression is frequently lost in human cancers. Here we
   analyzed the role of RECK in cervical carcinogenesis. Cervical cancer
   derived cell lines over expressing RECK were used to determine tumor
   kinetics as well as, cellular, immune and molecular properties in vivo.
   Besides, we analyzed RECK expression in cervical cancer samples. RECK
   over expression (RECK+) delayed tumor growth and increased overall
   survival in vivo. RECK+ tumors displayed an increase in lymphoid-like
   inflammatory infiltrating cells, reduced number and viability of tumor
   and endothelial cells and lower collagenase activity. RECK+ tumors
   exhibited an enrichment of cell adhesion processes both in the mouse
   model and cervical cancer clinical samples. Finally, we found that
   lower RECK mRNA levels were associated with cervical lesions
   progression and worse response to chemotherapy in cervical cancer
   patients. Altogether, we show that increased RECK expression reduced
   the tumorigenic potential of HPV-transformed cells both in vitro and in
   vivo, and that RECK down regulation is a consistent and clinically
   relevant event in the natural history of cervical cancer.

   Keywords: cervical cancer, HPV, tumorigenesis, MMP inhibitors, RECK

1. Introduction

   Worldwide, cervical cancer is the third most frequent and one of the
   ten most lethal neoplasias among women, accounting for over 565,000 new
   cases every year. Over 70% of these cases and more than 75% of
   associated deaths occur in developing countries [[40]1,[41]2]. Cervical
   cancer is etiologically associated with persistent infection with
   high-oncogenic risk Human Papillomavirus (HPV) types [[42]3].

   HPV-mediated carcinogenesis requires the sustained expression of viral
   E6 and E7 oncogenes that induce cell immortalization, resistance to
   apoptosis, evasion of innate and adaptive immune responses and
   alterations in the expression and activity of extracellular matrix
   (ECM) components [[43]4,[44]5,[45]6,[46]7]. The direct effect of HPV
   oncogenes on the expression of different ECM components has been
   previously reported. A recent study demonstrated that c-Jun inhibition
   in HPV-transformed cell lines (HeLa and CasKi) was associated with
   lower metalloproteinases type 9 (MMP-9) mRNA expression levels [[47]8].
   Furthermore, Shiau and coworkers showed that HPV16 E6 induced MMP-2 and
   -9 mRNA expression levels through an IL-8 dependent pathway in H1299
   cells [[48]9]. Finally, phosphorylation of HPV18 E7 at S32 and S34
   amino acids was associated with the up regulation of MMP-1 and -13
   protein expression in C4-1 cervical cancer cells [[49]10]. An extensive
   description of the main alterations in ECM components during
   HPV-associated transformation can be found elsewhere [[50]6].

   Up regulation of metalloproteinases type 2 and 9 (MMP-2 and MMP-9)
   expression and activity are the most common ECM related modifications
   in precursor cervical lesions and invasive carcinoma
   [[51]6,[52]11,[53]12,[54]13,[55]14]. We reported that this effect
   correlated with the expression of MMP inhibitors, including
   Reversion-inducing Cysteine-rich protein with Kazal motifs (RECK)
   [[56]13,[57]15]. RECK is a serine protease inhibitor that regulates
   various physiological events including the structural maintenance of
   ECM, correct formation of blood vessels during embryogenesis,
   organogenesis, tissue integrity, early neuronal differentiation and
   limb formation, with RECK deficiency being lethal in mouse embryos
   [[58]16,[59]17,[60]18]. Besides, reduced RECK expression has been
   associated with different pathologies, including cancer
   [[61]19,[62]20,[63]21,[64]22,[65]23,[66]24].

   We previously showed that RECK expression is down regulated in most
   cervical cancer derived cell lines and in cervical lesions
   [[67]13,[68]25]. Besides, we observed that HPV16 oncogenes expression
   was associated with reduced RECK protein levels in primary human
   keratinocytes cultures [[69]15]. Altogether, these observations
   indicate that RECK inhibition constitutes an important event in
   cervical carcinogenesis.

   In the present study, we show that RECK expression reduces de
   tumorigenic potential of HPV-transformed cells in vivo and alters the
   profile of the tumor inflammatory infiltrate. Besides, we show that
   RECK down regulation is an early trait in cervical cancer history and
   that RECK levels correlates with protease inhibitors expression,
   cervical lesions progression, presence of metastases and treatment
   response in clinical samples.

2. Materials and Methods

2.1. Cell Culture

   Cervical cancer derived cell line SiHa (HPV16 positive; ATCC^®
   HTB-35^TM, Manassas, VA, USA) and SW756 (HPV18 positive; ATCC^®
   CRL-10302^TM) were cultured in MEM medium supplemented with 10% fetal
   bovine serum (M10). Immortalized human embryonic kidney cells (HEK293T;
   ATCC^® CRL-3216^TM) were cultured in DMEM medium supplemented with 10%
   fetal bovine serum (D10). All cell culture procedures were performed at
   37 °C and 5% CO[2]. For cell proliferation analysis cells were seeded
   in 24 well plates (1.0 × 10^4 cells/well) and cultured for 1 to 8 days.
   Cells were counted every 24 h using a hemocytometer.

2.2. Obtention of Cells over Expressing RECK

   The lentiviral vector p156RRLsinPPCCMVIns3IRESPRC (pLV EGFP), described
   elsewhere, was used for RECK over expression [[70]26]. Lentiviral
   particles were obtained by transfecting packaging plasmids p59, p60 and
   p61 [[71]27] into HEK293T cells. For each transfection, 1.0 × 10^6
   cells were plated in 100 mm Petri dishes with 10 mL of D10 medium and
   incubated for 24 h. Then, 2.0 µg of each plasmid p59, p60 and p61 and
   2.0 µg of the lentiviral vector were added to 12 µL of Lipofectamine^TM
   3000 reagent (ThermoFisher, Waltham, MA, USA) and 16 µL of P3000^TM
   reagent supplied by the manufacturer. Transfection efficiency was
   monitored in a fluorescence microscope after 24 and 48 h (EVOS^®FL AMG,
   ThermoFisher Scientific, Waltham, MA, USA) for EGFP signal detection.
   After 48 and 72 h cultures supernatants containing the lentiviruses
   were collected and stored at −80 °C until use.

   For lentiviral transduction, 2.5 × 10^4 SiHa or SW756 were seeded in
   96-well culture plates with 200 μL of M10 medium for 24 h and
   transduced with 200 μL of lentivirus stock and polybrene (4 μg/mL)
   (Sigma-Aldrich, Saint Louis, MO, USA) for 24 h. Transduction efficiency
   was monitored after 24 and 48 h by EGFP signal detection in a
   fluorescence microscope (EVOS^®FL, AMG, USA). Finally, aseptic
   fluorescence-activated cell sorting (FACS) was performed in a cytometer
   (BD FACSaria^TM II U, San Jose, CA, USA) to isolate EGFP+ SiHa or SW756
   transduced cells ([72]Figure S1).

2.3. Western Blot

   Total protein extracts from monolayer cell cultures were prepared by
   lysis in RIPA buffer (150 mM NaCl, 50 mM Tris HCl-pH 7.5, 0.5% NP-40,
   0.1 mM EDTA) supplemented with protease inhibitors (Complete Mini,
   Roche Diagnostics).

   Altogether, 60 µg of total protein extracts were subjected to 10%
   SDS-PAGE polyacrylamide gel electrophoresis using the mini-Protean II
   Cell system (Bio-Rad, Hercules, CA, USA) and transferred to a
   polyvinylidene difluoride membrane (PVDF, Hybond-P, Cytiva, MA, USA).
   Protein expression was detected using anti-RECK protein (110 kDa, Cell
   Signaling CS3334, 1:1000 dilution), and anti-β-Tubulin (55 kDa,
   ThermoFisher, Waltham, MA, USA, 1:10,000 dilution) antibodies,
   horseradish peroxidase (HRP)-conjugated secondary antibodies and
   revealed using Enhanced Chemiluminescence procedures (Cytiva,
   Marlborough, MA, USA).

2.4. Clonogenic, Migration, Invasion and Anchorage Independent Growth Assays

   Cells were seeded in six-well plates (1.0 × 10^2 cells/well) and
   cultured in M10 medium for 15 days. For migration and invasion assays 5
   × 10^4 cells/chamber suspended in MEM were added to the upper
   compartments of the Transwells with 8 μm pores (Corning Incorporated,
   Corning, NY, USA). M10 was added to the wells for chemotactic
   attraction, followed by incubation of the plates for 12 h for migration
   and 36 h for invasion assay. Cell invasion was assessed by their
   ability to transpose both a Matrigel^® coat (BD Biosciences, San Jose,
   CA, USA) and barrier (Corning Incorporated, USA). Colonies, migrating
   and invading cells were fixed with 70% ethanol solution (Merck
   Millipore, Darmstadt, Germany), stained with 0.5% crystal violet
   (ThermoFisher, Waltham, MA, USA) in 10% ethanol solution and counted.
   For anchorage independent growth assay, 2.5 × 10^2 cells were suspended
   in 500 μL of M10 medium with 0.6% agarose and seeded in 24-well plates
   coated with the 1% agarose, and covered with M10 medium. After 30 days
   50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
   (MTT, 5mg/mL) was added for staining and counting of colonies.

2.5. Tumor Spheroids Formation

   Spheroid formation was assessed with culture of 5 × 10^3 (SiHa or
   SW756) seeded over 50 µL 1% agarose in PBS coat in U bottom 96 well
   plates. Spheroids were evaluated after seven days. Proliferation and
   cell viability were evaluated, after mechanical disruption, by cell
   count with Trypan blue 0.4% (ThermoFisher, Waltham, MA, USA) dye 1:10,
   using hemocytometer.

2.6. In Vitro Zymography

   Monolayer cell cultures were maintained in FBS free MEM for 72 h, when
   the supernatants were harvested and centrifuged at 1000× g for 10 min
   at 4 °C. Supernatants were stored at −80 °C until use. Zymography was
   performed according to DQ™ Collagen, type IV from Human Placenta,
   Fluorescein Conjugate protocol (Thermo Fisher Scientific, MA, USA) and
   as previously described [[73]28]. Briefly, 100 µL of FBS free
   supernatant was mixed with 100 µL of reaction solution (50 nM Tris, 5
   mM CaCl[2]-pH 7.5, 1 µM ZnCl[2] and 50 µg/mL DQ™ Collagen kept in 0.03%
   sodium azide) in black 96 wells plates. After two hours in the dark at
   37 °C the fluorescence was measured in Glowmax fluorimeter with a
   490/510 nm filter.

2.7. SDS-PAGE Gelatin Zymography

   Cell culture supernatants (30 µg) were subjected to non-denaturing
   electrophoresis in 5% gelatin SDS-PAGE in 65 V for 30 min followed by
   90 V for two hours at room temperature. After, the gel was washed twice
   with 2.5% Triton X-100 solution for 15 min at room temperature followed
   by 48 h of incubation with the reaction solution (50 nM Tris, 5 mM
   CaCl[2]-pH 7.5, 1 µM ZnCl[2]) at 37 °C. Then, the gel was stained using
   Coomasie brilliant blue R-250 0.5% for 5 min followed by sequential
   incubations with Methanol/Acetic acid (10%:10%) solution until bands
   were observed.

2.8. Tumor Growth Kinetics in Nude Mice

   All experiments involving mice were carried out under SPF conditions in
   the Isogenic Mice Animal Facility in the Department of Immunology at
   University of São Paulo. This project was approved by the Ethics
   Committee on the Use of Animals (CEUA, protocol number 138, page 13
   from book 03) of the ICB/USP. For tumor analysis 2 × 10^6 cells were
   injected s.c. on the dorsal flanks of female nude mice (10 animals per
   group). Tumor diameter was measured once a week with manual caliper and
   tumor volume was calculated using the formula V = (D × d^2)/2, where V
   is tumor volume, D is the largest diameter measured and d the smallest
   diameter measured. Tumors were allowed to grow up to 500 mm^3, when the
   animals were euthanized. Tumors were collected immediately and
   processed for the different types of analysis.

2.9. Analysis of Intratumoral Cell Populations

   Single cell tumor suspensions were prepared by mincing the tumors with
   a scalpel and digestion of the fragments in 1 mg/mL Collagenase I and
   IV diluted in Hank’s solution (15 mmol/L HEPES-pH 7.4, 0.5 U/mL DNase
   (Worthington, Columbus, OH, USA) and 5% fetal bovine serum) at 37 °C
   and 1300 rpm agitation. Cells were then filtered through a 70 μm
   strainer. Single cells (1 × 10^6) were suspended and labeled for
   surface antigens with specific antibodies (Antibodies references in