Interleukin 8 is a biomarker of telomerase inhibition in cancer cells

HCT116 cells were a kind gift of Bert Vogelstein (Johns Hopkins Medical School). COLO205 (ATCC® CCL-222™) and OVCAR5 cells (obtained as part of NCI-60 cell line panel, NCI) were obtained from American Type Culture Collection (ATCC) and were grown as recommended. Cells were treated with 2.5 μM imetelstat or mismatch oligonucleotide (Geron Corporation) twice per week for up to 6 weeks and did not exceed 80% confluence during the treatment. IL8 (GFP tagged) cDNA ORF was obtained from origene. Reagents RPMI 1640 (Catalog no. # 11879020, Thermofischer scientific), DMEM (Catalog no. # 11966092, Thermofischer scientific), Penicillin-streptomycin (Catalog no. # 15140122, Thermofischer scientific) and Fetal Bovine serum (Catalog no. # 10082147, Thermofischer scientific) were purchased from Thermofischer scientific.

IL8 and control non-specific shRNAs were obtained from Open Biosystems. Table 1 shows the product IDs for all shRNAs. Lentiviral particles were prepared by co-transfecting the shRNA plasmids and lentiviral packaging plasmids, pSPAX2 and pMD2.G, into 293 T cells using Effectene (Qiagen) and following the protocol at the Broad Institute’s website (http://​www.​broadinstitute.​org/​rnai/​public/​resources/​protocols).

To measure cell viability two kinds of assays were performed. For Trypan blue exclusion viability assays, cells were plated and were treated with mismatch oligo’s or imetelstat for 2 weeks triplicate. After 2 weeks of treatment they were trypsinized and were with mixed with an equal volume of Trypan Blue Solution (Invitrogen) and counted using Countess (Invitrogen). Relative cell viability of imetelstat treated cells with respect to control mismatch oligo treated cells is plotted. For MTT assay cells are plated in in 96 well plate. Then they were either treated with mismatch oligo’s or imetelstat for 2 weeks. After treatment, 10μl of 5 mg/ml of MTT is added to the medium each well and incubated for 1 h. Medium is removed and 100μl of DMSO is added, mixed well and the measurement is performed at absorbance 590 and 630 nm wavelength. Average is taken and the reading at 690 is subtracted form that at 590. Relative cell viability of imetelstat treated cells with respect to control mismatch oligo treated cells is plotted. For colony formation assays, 10³ cells were seeded in triplicate. Then they were either treated with mismatch oligo’s or imetelstat for 2 weeks. Colonies formed were stained with 0.005% crystal-violet solution and counted. For in vivo experiment, Eight-week old, athymic nude (NCr nu/nu) mice (n = 5) were injected subcutaneously with cancer cells (2.5 × 10⁶). After one week, tumor-bearing mice received mismatch oligonucleotide or imetelstat (30 mg/kg bodyweight) three times per week by intraperitoneal injection. Tumor growth was measured every week using calipers, and tumor volumes were calculated using the formula 0.5 X length X width² for every week.

qRT-PCR and Immunoblot was performed as described in [20]. Briefly For mRNA expression analyses, total RNA was extracted with TRIzol (Invitrogen) and purified using RNAeasy mini columns (Qiagen), and cDNA was generated using M-MuLV first-strand cDNA synthesis kit (New England Biolabs) as per manufacturer’s instructions. Quantitative RT-PCR was performed using Power SYBR-green kit (Applied Biosystems) for mRNA expression analysis or the miScript SYBR-green PCR assay kit (Qiagen), as per manufacturer’s instructions. Actin was used as an internal control. For Immunoblotting whole cell protein extracts were prepared using IP lysis buffer (Pierce) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Sigma-Aldrich, St.Louis, MO). Protein concentration was estimated using a Bradford Assay kit (Bio-Rad). Proteins separated on 10% or 12% polyacrylamide gels were transferred to PVDF membranes using a wet transfer apparatus from Biorad. Membranes were blocked with 5% skim milk and probed with primary antibodies followed by the appropriate secondary HRP-conjugated antibody (GE healthcare, UK). Blots were developed using the Supersignal Pico Reagent. Antibody and primer information is provided in Table 1.

The TRAP assay was performed essentially as described [23]. Every plate included standards, inactivated samples and lysis buffer as controls. Each sample was analyzed at least in triplicate. Telomerase activity was plotted relative to control mismatch oligonucleotide treated cells. Telomere length measurement was performed using Relative Human Telomere length Quantification qPCR assay kit from Science cell (Catalog no. # 8908). The assay was performed as described by the supplier.

For microarray experiments using HCT116, OVCAR5 and COLO205 (5 million) cells were treated with imetelstat for 2 weeks, total RNA was isolated from the cells as described above and used to generate labelled antisense RNA. All antisense RNAs were made using the Ambion MessageAmp Kit and hybridized to Illumina HumanHT-12 V4.0 expression BeadChip using Illumina’s protocol.

To identify biomarker of telomerase-inhibitor therapy, we analyzed a series of cancer cell lines of different tissue origin (Table 2). To this end, we treated these cancer cell lines, with imetelstat, a telomerase inhibitor. Imetelstat is a synthetic lipid-conjugated, 13-mer oligonucleotide N3’ P5’-thio-phosphoramidate and is complementary to the template region of telomerase RNA (hTR). Inside the cells, imetelstat acts as a competitive enzyme inhibitor that binds and blocks the active site of the enzyme (a “telomerase template antagonist”) thus inducing growth inhibition [24, 25].

We first optimized the imetelstat concentration for the treatment of various cancer cell lines. To do so, we treated different cancer cell lines with multiple concentrations of imetelstat for determining IC50. We found that IC50 for imetelstat for different cancer cell lines was in the range of 2.5μM as shown in Additional file 1: Figure S1. Next, we treated multiple ovarian and colon cancer cell lines of different tissue origin for 2 weeks with 2.5μM imetelstat and identified HCT116, COLO205 and OVCAR5 as three cancer cell lines that showed strong growth inhibition following imetelstat treatment and also showed concomitant decrease in telomerase activity (Fig. 1). We additionally checked telomere length in the cell lines that showed growth inhibition upon imetelstat treatment and found that HCT116, COLO205 and OVCAR5 show shortened telomere length upon imetelstat treatment as compared to control treated cells (see Additional file 2: Figure S2). These results demonstrate that inhibition of telomerase in ovarian and colon cancer cell lines leads to their growth attenuation.

In order to identify the genes that can function as biomarker to predict response to telomerase-based therapy, HCT116, COLO205 and OVCAR5 cell lines that showed strong growth inhibition following imetelstat treatment were treated with imetelstat for 2 weeks and then transcriptome-wide gene expression analysis was performed (Fig. 2a). We observed multiple genes that were upregulated and downregulated in each cancer cell line (Fig. 2b). We also performed analysis to identify the common genes that were altered upon imetelstat treatment in all these cancer cell lines. Our results showed that in all three cancer cell lines, IL8 was the only common candidate that was downregulated in imetelstat treated cells as compared to control cells (Fig. 2b). These results in sum indicate that the cancer cells that show response to telomerase inhibition downregulate IL8 levels and this phenomenon is of general occurrence as multiple ovarian and colon cancer cell lines that undergo growth inhibition upon imetelstat shows IL8 downregulation (Fig. 2b).

In Fig. 1, we have shown the effect of imetelstat on cancer cell viability using trypan blue exclusion assay. In order to confirm the growth inhibitory effect of imetelstat on HCT116, COLO205 and OVCAR5 cells we employed an additional assay called MTT assay. MTT is a calorimetric based assay to detect metabolic activity of cell which is dependent of the number of viable cells. To do so, we treated HCT116, COLO205 and OVCAR5 cells with imetelstat for 2 weeks and then performed MTT assay as described in method section. As expected, we found that after two weeks of imetelstat treatment HCT116, COLO205 and OVCAR5 cells showed significant growth inhibition (Fig. 3a). This was further confirmed by colony formation assay as shown in Fig. 3b. Next, in order to study the effect of imetelstat on the growth of these cancer cell lines in vivo, we performed xenograft-based mouse tumorigenesis assay. Our results showed that imetelstat significantly inhibited the growth of HCT116, COLO205 and OVCAR5 tumors upon imetelstat treatment (Fig. 3c). These results confirmed that the inhibition of telomerase affects the cancer cell growth. Therefore, expression of telomerase is critical for cancer cell survival and serves as an effective target to inhibit cancer cell growth.

After confirming the cancer cells growth suppression upon telomerase inhibition, we measured IL8 transcript and protein level in HCT116, COLO205 and OVCAR5 cells after imetelstat treatment and found that both IL8 mRNA as well as IL8 protein level decreases upon imetelstat treatment (Fig. 4). This was observed in multiple ovarian and colon cancer cell lines suggesting that decrease in IL8 level is a common biomarker predictive of telomerase inhibition induced cancer cells growth suppression (Fig. 4). We also checked IL8 levels in the cell lines that did not show growth suppression upon telomerase inhibition and found that in these cell lines IL8 level did not change upon imetelstat treatment, as shown in Additional file 3: Figure S3. These results in sum indicate that decrease in IL8 level is a specific biomarker for the cancer cell lines that shown growth suppression upon telomerase inhibition.

Our result showed that IL8 level decreases upon imetelstat treatment. To confirm if the decrease in IL8 levels leads to decrease in cell viability, we knocked down IL8 expression using shRNAs in different cancer cell lines (Fig. 5a). Next, these cells were checked for cell viability. As shown in Fig. 5b, we find that the cells expressing IL8 shRNA have lower cell viability as compare to control cells expressing non-specific shRNA. In order to conclusively show the inhibition of IL8 is involved in telomerase inhibition induced growth inhibitory effect, we overexpressed IL8 in imetelstat treated cells (Fig. 5c), and then checked the cell viability. We found that IL8 overexpression rescued telomerase inhibition induced growth inhibitory effect (Fig. 5d). This was not due to restoration of telomerase activity upon IL8 expression, because no change in telomerase activity was observed after IL8 over expression in imetelstat treated cells (Fig. 5e). Taken together, these results led us to conclude that telomerase inhibition leads to decreases IL8 levels, which can be employed as a biomarker for predicting response to telomerase-based therapy in cancer.