Tumor suppressive effects of WEE1 gene silencing

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Original Article

Tumor suppressive effects of WEE1 gene silencing could not enhance immunopotentiation effects of CD80 and 4‑1BBL co‑stimulation in human T cells ABSTRACT Background: Activation of T cells against tumors by recruiting co‑stimulatory molecules has been an attractive approach for cancer immunotherapy. Reports suggested that targeting different genes in tumors might also boost T cell‑mediated tumor destruction. Aims: We investigated whether in vitro WEE1 gene silencing in MDA‑MB‑468 and MCF7 breast cancer cell lines could enhance immunopotentiating effects of CD80 and 4‑1BBL co‑stimulation in human T cells. Materials and Methods: WEE1 gene was specifically silenced in the cancer cells using shRNA technology. The co‑stimulatory molecules were over‑expressed on the surface of the cancer cells by recombinant non‑replicative adenoviruses. The immune reaction of T cells in the co‑culture with tumor cells was studied. IFN‑γ production was assessed by intracellular staining of T cells. To assess cytotoxic activity of CD8+ T cells, the CD107a mobilization‑degranulation assay was performed. Expression of granzyme B, perforin and fasl were examined by real time PCR. Results: T cell dual co‑stimulation led to a significant increase in the frequency of IFN‑γ producing cells and higher percentages of degranulation in CD8+ T cells. It also resulted in higher expression levels of the cytotoxicity‑related genes. WEE1 gene silencing in the target cells alone however, could not produce significant immune reactivation in the cultured T cells. Likewise, the immune responses of T cells neither improved nor suppressed when dually co‑stimulated PBMCs were exposed to the cancer cells with silenced WEE1. Conclusions: In spite of antitumor effects of WEE1 silencing, combination of this approach with immune co‑stimulation could not boost the reactivity of cultured T cells against the tested breast cancer cells. KEY WORDS: 4‑1BBL, breast cancer cell, CD80, co‑stimulation, immunopotentiation, T cell, WEE1

INTRODUCTION Despite the fact that the immune system recognizes tumor antigens and reacts against tumors, in many cases tumors continue to grow and escape from rejection.[1] The literature shows several escape mechanisms that have been used by different tumors. Among those, induction of anergy in tumor‑specific T cells by poor expression of co‑stimulatory molecules has been considered as one of possible mechanisms.[2] It sabotages the immunogenicity of tumor antigens and dampens the immune reaction. Hence, activation of T cells against some tumors by recruiting co‑stimulatory molecules has become an attractive approach for cancer immunotherapy.[3] Among the many known ligands and receptors, several molecular pairs have been described to work as T cell co‑stimulators. Signals through CD28 (binds to CD80) and 4‑1BB (binds to 4‑1BBL)

(CD137/CD137L) co‑stimulate T cells during early and late activation, respectively. Different reports in the literature have demonstrated the roles of these two co‑stimulatory pathways in priming and recall responses of T cells to Ags and tumors.[4,5] Moreover, it has been shown that combination of CD80 and 4‑1BB signals could increase the magnitude and duration of the immune response.[6] Interestingly 4‑1BB signal could reactivate T cells after the cells became unresponsive to signals through CD80/CD28 interaction.[7] Such findings hold promise for the application of these co‑stimulatory molecules in cancer immunotherapy. Some preclinical studies have shown that the adoptive transfer of ex vivo 4‑1BB and CD28 co‑stimulated T cells could induce antitumor immune response against poorly immunogenic cancers.[8,9] The benefits of these co‑stimulatory molecules have been exploited to produce artificial APCs (aAPCs) and efficiently stimulate antigen‑specific T cells ex vivo.[10]

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Naghmeh Ghiasi1,3, Mojtaba Habibagahi2, Rozita Rosli3,4, Abbas Ghaderi1,2, Khatijah Yusoff4,5, Ahmad Hosseini1, Syahril Abdullah3, Mansooreh Jaberipour1 School of Medicine, Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences, 2Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran, 3Genetics and Regenerative Medicine Research Centre, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, 4 UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, 5 Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor DE, Malaysia 1

For correspondence: Dr. Mansooreh Jaberipour, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran. E‑mail: jaberim@ sums.ac.ir Access this article online Website: www.cancerjournal.net DOI: *** PMID: *** Quick Response Code:

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Ghiasi, et al.: No extra immune enhancement by WEE1 silencing in tumor cells

Beside these, new methods of cancer‑targeted therapies have been able to tackle cancer cells by interfering with tumor growth, carcinogenesis or metastasis.[11]

China) supplemented with 10% fetal bovine serum and contained 100 unit/ml penicillin and 100 µg/ml streptomycin. Cultures were incubated at 37°C with 95% humidified air and 5% CO2.

Interestingly, there are accumulating experimental evidence that targeted therapies can also stimulate antitumor immune response by various mechanisms. They can decrease tumor‑induced immune suppression through blocking the production of immune suppressive molecules such as VEGF[12] and TGF‑β,[13] or may up‑regulate the immune regulatory molecules including MHC,[14] co‑stimulatory and adhesion molecules[15] on the surface of tumor cells. Targeted therapies may also exert their immune‑enhancing action by induction of a form of immunogenic cell death in cancer cells.[16] These positive interactions between targeted therapy and immunotherapy have opened a new era for cancer treatment. There has been great interest lately in the combination of anti‑tumor effects of targeted therapies with the immune active compounds to provide a more potent anti‑tumor response. This concept has emerged based on promising preclinical and clinical studies.[17,18] However, more combinations should be evaluated to find the best choice for each type of tumors although that cannot be the only choice. In that regard, investigations are ongoing to find suitable targets for different tumors.

A total of 10 healthy volunteers (mean age; 35 ± 5) were enrolled in the study. All participants gave their informed consent. The study was approved by the ethics committee of the University. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll‑Hypaque density gradient centrifugation and depleted from plastic‑adherent cells. The isolated PBMCs were cultured in complete media and stimulated with a suboptimal concentration of 100 ng/ml soluble OKT3 anti‑CD3 monoclonal antibody (Janssen‑Cilag, Buckinghamshire, UK).

WEE1 kinase is a key molecule in maintaining G2‑cell‑cycle checkpoint arrest for pre‑mitotic DNA repair. It is over‑expressed in various cancer types, including breast cancer.[19] Therefore, inhibition of WEE1 could be a promising strategy. It has been shown that inhibition of WEE1 could induce caspase‑mediated apoptosis and decreases tumor volume in vivo in medulloblastoma.[20] In our previous study, in concordance with others, we showed that WEE1 inhibition could significantly decrease the viability of breast cancer cells; induce G2 arrest abrogation and increase apoptotic cells (sub‑G1). Moreover, the observed interactions between the pro‑ and anti‑apoptotic proteins and decrease in the angiogenesis marker (VEGF) expression confirm the susceptibility to cell death and validate the tumor suppressive effect of WEE1 inhibition in MCF7 and MDA‑MB‑468 breast cancer cells.[21,22]

Expression of co‑stimulatory ligands The E1‑ and E3‑deficient recombinant non‑replicative adenoviruses expressing CD80 (Ad‑CD80) and 4‑1BBL (Ad‑4‑1BBL) were courtesy provided by Dr. P. F. Searle, from the Gene Therapy Laboratory, Birmingham University, UK. To express the transgenes, MCF7 and MDA‑MB‑468 breast cancer cell lines were co‑infected with 300 virus particles (vp) per cell of the viruses. As the control experiment, cells were infected similarly with 600 virus particles (vp) per cell of adenoviral expressing enhanced green fluorescent protein (Ad‑GFP). The infected cells were kept for 48 hours to reach to the maximum transgene expression before adding purified PBMCs into the cultures. The expressions of the transgenes were assessed using flow cytometry analysis.

MATERIALS AND METHODS

Co‑culture of PBMCs with gene‑modified breast cancer cells MCF7 and MDA‑MB‑468 breast cancer cell lines were infected with recombinant viruses and distributed into 24 well plates at 1 × 105 cell/ml to form monolayers. To investigate whether WEE1 gene silencing in cancer cells can enhance the immune potentiating by CD80 and 4‑1BBL co‑stimulation, gene specific shRNA were used to suppress the expression of the gene 24 hours post‑infection. To this end, a pool of four WEE1 specific shRNA plasmids and a non‑targeted scrambled type were used to suppress the WEE1 gene or as control, respectively. shRNAs were transfected into the cells using lipofectamin 2000 (Invitrogen, Grand Island, New York, USA) according to the manufacturer’s instruction with some modifications as described elsewhere. The sequence of each 29mer shRNA construct and the target locations have been also listed.[21] The purified PBMCs at 1 × 106 cell per well were added to the wells and stimulated with suboptimal concentration of 100 ng/ml of soluble OKT3 anti‑CD3 monoclonal antibody (Janssen‑Cilag, UK). Cultures were incubated for a further three days and then the downstream experiments for the measurement of the reactivity of the T cells was performed.

Cell lines and peripheral blood mononuclear cells MDA‑MB‑468 and MCF7 cell lines were purchased from the National Cell Bank of the Institute Pasteur of Iran. The cell lines were cultured in RPMI‑1640 medium (Sigma‑Aldrich, Hong Kong,

Intracellular interferon gamma cytokine detection in cultured PBMCs The night before the assays, the PBMCs in the co‑culture with cancer cells were restimulated with 100 ng/ml

In the present study, we designed in vitro models to investigate whether WEE1 gene targeting in breast cancer cell lines could enhance the immune potentiating effects of CD80 and 4‑1BBL and intensify T‑cell responses. The results of this study may contribute towards better understanding of the effectiveness of this combination strategy in breast cancer treatment where each individual modality has its own advantages and disadvantages.

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Ghiasi, et al.: No extra immune enhancement by WEE1 silencing in tumor cells

of anti‑CD3. To inhibit the secretion of the cytokines, brefeldin‑A (1 µl/ml BD, CA, USA) was added into the cultures during the last five hours of the culture time. PBMCs were harvested, washed with PBS/FBS (2%) and stained with fluorescent conjugated monoclonal antibodies against CD4 (APC conjugated, BD Pharmingen, CA, USA) or CD8 (Percp conjugated, BioLegend, CA, USA). Intracellular interferon gamma [IFN‑γ] was stained with PE conjugated anti‑h IFN‑γ antibody (BD Pharmingen, CA, USA) after fixation (PBS 1x, 1% paraformaldehyde, 0.1% sodium azide) and permeabilization (PBS 1x, 0.2% saponin, 0.1% sodium azide, 4% FBS). To exclude any non‑specific binding, isotype matched control antibodies were used. Cells were analyzed by FACSCalibur flow cytometer (BD Pharmingen™, CA, USA) using CellQuest pro (BD Biosciences) as acquisition software. Data were reanalyzed and graphically presented by FlowJo software (Tree Star, Inc, USA). CD107a mobilization – degranulation assay The breast cancer cell lines were transduced with the recombinant adenovirus vectors and transfected with the shRNAs (pool of four WEE1 or scrambled) as previously explained. PBMCs were isolated and activated by membrane‑bound anti‑CD3 antibody (clone OKT3) during an overnight incubation in flasks at 2 × 106 cell/ml. The activated cells were harvested, washed with PBS/FBS (2%) and stained with APC‑conjugated anti‑CD107a monoclonal antibody (BioLegend, CA, USA) at the appropriate concentration. Subsequently, the stained cells were transferred into the wells that contained cancer cells with or without the expression of the co‑stimulatory genes and shRNA treatment. The cells were incubated for one hour and then GolgiPlug and GolgiStop (BD, CA, USA) at a final concentration of 10 µg/mL and 6 µg/mL, respectively, were added to the cultures and incubated for additional 5 hours. For the positive control, PBMCs were activated with phorbol‑12‑myristate‑13‑acetate (PMA) (50 ng/mL) and inomycin (250 µg/mL). At the end of the incubation period, the cultured PBMCs were harvested and stained with Percp conjugated anti‑CD8 (BioLegend, CA, USA). The cells were then fixed, permeabilized and stained intracellularly with PE conjugated anti granzyme B antibody (BD Pharmingen, CA, USA) and analyzed by flow cytometry. Quantitative real‑time PCR After three days of PBMCs co‑culture with cancer cells in different experimental conditions, the PBMCs were harvested and total RNA was extracted from the cells using TRIzol reagent (Life Technology, CA, USA). Reverse transcription to cDNA was performed using RevertAidTM H‑Minus First Strand cDNA Synthesis Kit (Fermentas, Helsinki, Finland). The relative quantity of perforin, granzyme B and fasl transcripts were measured against 18s rRNA as housekeeping gene using ABI 7500 thermocycler machine (Applied Biosystems Applied, CA, USA). Sequences of used primers and probes are listed in Table 1. The results from the uninfected culture conditions Journal of Cancer Research and Therapeutics - ??? - Volume ?? - Issue ?

were regarded as reference for all calculations. The 2‑ΔΔCT equation was applied to the data to calculate the expression of the genes of interest. Statistical analysis The differences between the experimental and the control conditions were analyzed by Wilcoxon signed rank test by GraphPad Prism version 5 software (GraphPad Software, CA, USA). In all statistical analysis, P