Identification of telomerase inhibition responsive Cancer cell lines

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].

Table 1 Primer sequences for RT-qPCR analysis; clone ID and catalog numbers for shRNAs (Open Biosystems); antibodies used Full size table

Table 2 Cell lines and their tissue origin Full size table

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.

Fig. 1 Measurement of response of cancer cells to telomerase inhibition. Indicated cell lines were treated with mismatch oligonucleotide nucleotide or imetelstat for 2 weeks. a Cell viability was monitored by trypan blue exclusion assay and relative cell viability with respect to mismatch oligonucleotide nucleotide treated cell is plotted (b) Telomerase activities was measured by TRAP assay and relative telomerase activity with respect to mismatch oligonucleotide nucleotide treated cell is plotted. Error bar shows Standard Error Mean (SEM). (**, p < 0.001), (ns, non-significant) Full size image

Transcriptome-wide gene expression analysis identifies IL8 as a biomarker that predict response to telomerase inhibition

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).

Fig. 2 Gene expression analysis using Illumina identified IL8 as a common biomarker of telomerase response. a Schematics of the Transcriptome-wide gene expression analysis is described. It was performed in HCT116, OVCAR5 and COLO205 cells. Briefly equal number of cells from these cell lines were either untreated or treated with imetelstat for 2 weeks after which RNA was isolated and candidate genes that were either upregulated or downregulated were identified. b Venn diagram showing the common genes that were identified after imetelstat treatment in between two cell lines and also between three cell lines Full size image

Telomerase inhibition suppresses cancer cell viability

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.

Fig. 3 Telomerase inhibition suppresses cancer cell viability. a Cell viability was measured by MTT assay. Relative cell viability is plotted in 2 weeks imetelstat treated cells in comparison to control mismatch oligonucleotide treated cells for the shown cell lines. b Indicated cell lines were treated with mismatch oligonucleotide or imetelstat for 6 weeks and stained with crystal violet. Representative wells are shown. c HCT116, OVCAR5 and COLO205 cells were injected subcutaneously in athymic nude mice. These were either treated with either mismatch oligonucleotide or imetelstat for 4 weeks. Average tumor volumes for indicated cell lines after 4 weeks of treatment are shown. Error bar shows Standard Error Mean (SEM). ** represents p < 0.001 Full size image

Telomerase inhibition decreases both IL8 mRNA and protein level

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.

Fig. 4 IL8 levels decrease upon inhibition of telomerase. Indicated cells were treated with imetelstat for 2 weeks. a IL8 RNA level was measured by qRT-PCR. Actin was used as internal control. Relative levels with respect to control mismatch oligonucleotide treated cell is plotted. b IL8 protein level was measured by immunoblotting. Actin was used as loading control. Error bar shows Standard Error Mean (SEM). (*, p < 0.01) Full size image

IL8 inhibition affects the cell viability and its overexpression rescues telomerase inhibition induced growth inhibitory effect

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.