HDAC11 levels are elevated in lung cancers and it correlates with poor prognosis

Experiments were conducted to assess the potential role of HDAC11 in NSCLC. An immunohistochemical analysis was carried out on human tissue microarrays to assess whether the levels of HDAC11 were altered in lung cancer. We found that the HDAC11 levels were elevated by ~2.5 fold in the lung adenocarcinoma as well as in squamous cell carcinoma as compared to normal lung tissue (Fig. 1A). Quantitation of the staining showed a significant (3 fold) increase in the expression of HDAC11 in metastatic NSCLC of both histological subtypes (Fig. 1B). The HDAC11 staining was also conducted on a second human tissue microarray. The quantitation of the HDAC11 staining in this TMA also showed a significant (3–4 fold) increase in HDAC11 expression in NSCLC tissues as compared to normal lung (Fig. 1C). Since we found an increase in HDAC11 expression in lung cancer, we examined whether the expression of HDAC11 predicts the prognosis of lung cancer patients. A Kaplan Meier survival analysis conducted on the publicly available KM Plotter dataset24 (http://kmplot.com/analysis/index.php?p=service&cancer=lung) using Probeset 219847_at showed that higher expression of HDAC11 correlated with poor survival in patients with lung adenocarcinoma as well as squamous cell carcinoma (Fig. 1D,E). Additional multivariate analysis of the data showed poor survival in early stage (Stage 1) lung adenocarcinoma patients with higher expression of HDAC11 (Supplementary Fig. 1A). Also, it was found that higher expression of HDAC11 in male lung adenocarcinoma patients correlated with poor survival (Supplementary Fig. 1B). Such a correlation was not observed in the female patients (data not shown); the reason for this disparity is not clear at this time. While this data was not evident when another probe set was tested, analysis of the Cancer Cell Line Encyclopedia showed that HDAC11 is overexpressed or amplified in 8% of cancer cell lines (data not shown). Similarly, an analysis of the Oncomine database suggested that there are variations in the levels of HDAC11 in tumors indicating that it might depend on the tumor histology or grade of the tumors.

Figure 1 HDAC11 expression in human lung tumor tissues and cells and its correlation with patient prognosis. (A) Elevated HDAC11 staining is seen in NSCLC tissue and its metastatic sites as compared to the normal human lung tissue in TMA. (B) Quantitation of IHC performed on the TMA shows a ~2.5–3-fold increase in HDAC11 expression in human lung adenocarcinoma tissue, squamous cell carcinoma (SCC) and metastatic lung carcinoma as compared to normal lung tissue. (C) Quantitation of second IHC staining for HDAC11 also showed 3-fold increase in HDAC11 expression in NSCLC patient tumor tissue as compared to normal lung tissue. (D,E) Kaplan-Meier survival analysis for HDAC11 (Probeset 219847_at) shows poor prognosis in lung adenocarcinoma (D) and squamous cell carcinoma (E) patients with higher mRNA expression of HDAC11 in them. (F) HDAC11 could be detected in multiple cell lines of different histology by a western blot analysis. Similarly, Sox2 and YAP1 could also be detected in these cells. The images of the full scan western blots are provided in Supplementary Fig. 6. Full size image

The expression pattern of HDAC11 in lung cancer cell lines was next examined. Western blot analysis was conducted on lysates from lung adenocarcinoma cells, immortalized tracheobronchial cells (AALE) and primary lung cancer associated fibroblasts (CAFs) (Fig. 1F). A549, H358 and H460 cells harbor a Kras mutation and H1650, HCC827 and PC9 cells carry mutation in the Egfr gene. Sox2, which is known to promote stemness of lung adenocarcinoma and is often amplified in squamous cell carcinoma, was expressed in these cells to varying levels15,18,25,26. YAP1, a transcriptional co-activator and the effector protein of hippo signaling pathway18,27, was expressed in these cell lines as well (Fig. 1F). Next, we assessed if expression of HDAC11 correlated with the expression of Sox2 in the tissues of lung cancer patients. Immunohistochemical analysis was carried out for HDAC11 and Sox2 on human lung TMAs (Supplementary Fig. 1A,B). HDAC11 expression was higher as observed earlier (Supplementary Fig. 1C). Our earlier publications have shown higher expression of Sox2 in NSCLC patient tumors17,18,19. The results here showed similar higher expression of Sox2 in NSCLC patients (Supplementary Fig. 1D). Pearson correlation coefficient analysis of the semi quantitative scores of HDAC11 and Sox2 showed a moderately positive correlation (r = 0.5041) (Supplementary Fig. 1E). These results suggested that HDAC11 was expressed in lung cancer tissues and cell lines and elevated levels of this protein might contribute to poor patient survival.

HDAC11 is elevated in cancer stem-like cells from lung adenocarcinoma cell lines and regulates Sox2

Recent reports suggest a possible role of HDACs in maintenance of CSCs, opening a new avenue to target these cells28. Since no information is available on the expression of HDACs in stem-like cells from NSCLC, we examined the levels of HDAC11 in Hoechst negative stem-like side population (SP) of NSCLC cell lines. We had found that the SP cells isolated from lung tumor explants as well as NSCLC cell lines have stem-like properties. SP cells could self-renew, were drug resistant and could transdifferentiate into CD31-positive angiogenic tubules17,18. A RT-PCR analysis showed that there was 1.5 to 3.5-fold increased expression of HDAC11 mRNAs in SP cells from A549 (Fig. 2A) and H1650 (Fig. 2B) cell lines, as compared to the non-stem main population (MP) cells. SP cells were found to have higher expression of ABCG2 mRNA, which was used as a control (Fig. 2A,B)17,18. Since we had found that multiple embryonic stem cell transcription factors are elevated in stem-like SP cells, we examined if HDAC11 regulates the expression of any of these factors. Towards this purpose, we depleted HDAC11 by siRNAs in A549 and H1650 cells and analyzed the expression of stem cell transcription factors Sox2, Oct4 and Nanog. Additional HDACs like HDAC1 and HDAC6 were also depleted as they were shown to have a role in self-renewal in embryonic stem cells and regulation of Sox2 in cancer respectively29,30. Depletion of HDAC1 or HDAC11 resulted in a reduction in the level of Sox2 mRNA in both A549 and H1650 cells (Fig. 2C,D respectively). However, there was no reduction in the expression of Oct4 and Nanog transcription factors upon the depletion of HDAC1, HDAC6 or HDAC11 (Fig. 2C,D). The depletion of HDAC1, HDAC6 and HDAC11 genes was also confirmed by RT-PCR in A549 and H1650 (Fig. 2C,D).

Figure 2 HDAC11 is elevated in stem-like side population (SP) from NSCLC cell lines and regulates Sox2. (A,B) Real time PCR analysis of mRNA from SP and MP cells of A549 (A) and H1650 (B) cell lines reveal significantly higher levels of HDAC11 mRNA in sorted SP cells. ABCG2 mRNA expression is used as positive control for SP cells. (C,D) Real time PCR analysis of cells depleted of HDAC1, HDAC6 or HDAC11 using siRNA transfections in A549 (C) and H1650 (D) cells show decrease in Sox2 mRNA expression as compared to control siRNA treatment. There was no change in Oct4 and Nanog expression. HDAC1, HDAC6 and HDAC11 mRNA expression confirmed the depletion status in both cell lines. (E) Transient transfection experiments in A549 and H1650 cells with Sox2 core promoter luciferase (Sox2-luc) construct and increasing concentrations of HDAC11 shows increasing promoter luciferase activity. (F) Co-transfection experiments of Sox2-luc and HDAC11 with YAP1 and Gli1 expression vectors showed an additive effect on Sox2-luc activity in both A549 and H1650 cells. Full size image

We then investigated if HDAC11 could induce the Sox2 promoter activity using transient transfection assays. The results showed that Sox2 promoter luciferase (Sox2-luc) activity increased in a dose dependent manner with increasing amounts of HDAC11 (Fig. 2E). Our earlier studies had shown that the hippo signaling pathway effector protein YAP1 and the hedgehog pathway transcription factor Gli1 could induce Sox2 transcription18,19; experiments were conducted to assess if HDAC11 could enhance the induction of Sox2-luc by YAP1 and Gli1. Transient transfections revealed that co-transfection of HDAC11 had an additive effect on the induction of Sox2-luc promoter by YAP1 or Gli1 (Fig. 2F). These results suggested that HDAC11 might act as downstream effector of the hippo or the hedgehog signaling cascades to induce genes involved in stemness.

HDAC11 interacts with Gli1 and regulate Sox2 expression

As our results showed that HDAC11 could induce Sox2 promoter activity along with Gli1 transcription factor, we examined if HDAC11 interacted with Gli1 using double immunofluorescence assays. We found that Gli1 and HDAC11 co-localized in both A549 and H1650 cells (Fig. 3A), suggesting that their physical interaction contributes to the induction of Sox2. This result was confirmed by an immunoprecipitation-western blot experiment, which showed the presence of Gli1 in HDAC11 immunoprecipitates (Supplementary Fig. 2). Our earlier report showed that drug resistance in cells lead to increased Sox2 protein expression19. Western blot analysis confirmed that higher Sox2 expression in gefitinib resistant PC9-GR cells as compared to parental PC9 cells (Fig. 3B). Similar results were also obtained in erlotinib resistant HCC827-ER cells as compared to parental HCC827 cells (Fig. 3B). Both the resistant cells also had higher levels of HDAC11, while there was no marked change in the levels of Gli1 (Fig. 3B). We next used these cells in chromatin immunoprecipitation (ChIP) assays and found that HDAC11 could be detected on the Sox2 gene promoter at the Gli1 transcription factor binding sites19 (Fig. 3C,E). The interaction was found to be higher in the resistant cells as compared to the parental cells in both HCC827/HCC827 ER and PC9/PC9 GR pairs. Acetylated histone H3 was used as positive control and IgG was used as negative control in this experiment (Fig. 3C,E). Myc promoter was used as overall negative control (Fig. 3D,F). Since Sox2 plays a significant role in the maintenance and survival of CSCs from lung adenocarcinoma cells17,18,19, we carried out the ChIP assays in stem-like side population (SP) cells isolated from H1650 lung adenocarcinoma cells. Our results showed presence of HDAC11 on Sox2 promoter region on Gli1 binding sites in the H1650 SP cells (Fig. 3G). Myc was used as negative control here as well (Fig. 3H). Taken together, these results suggest that HDAC11, in association with Gli1, associates with the Sox2 promoter to induce its expression.

Figure 3 Sox2 gene expression is regulated by Gli1 and HDAC11. (A) Double immunofluorescence assay with Gli1 and HDAC11 showed co-localization of both in A549 (upper panel) and H1650 (lower panel) cells. DAPI was used to stain the nucleus of the cells (B) Western blot analysis with PC9 parental and gefitinib resistant PC9 GR cells showed an increase in Sox2 and HDAC11 expression in the resistant cells. Increased Sox2 as well as HDAC11 proteins were also found in HCC827 erlotinib resistant (ER) cells as compared to the HCC827 parental cells. (C,D) A ChIP analysis in PC9/PC9 GR cells showed presence of HDAC11 with Gli1 binding site on Sox2 promoter (C) and this interaction was higher at the promoter in the resistant cells. Such a binding was not seen in the Myc promoter (D). (E,F) Similar ChIP analysis in HCC827/HCC827 ER cells showed increased association of HDAC11 with Gli1 at the Sox2 promoter in the erlotinib resistant HCC827 cells (E). No change was observed in the Myc promoter that was used as a control. (G,H) The ChIP analysis on H1650 side population cells showed the interaction of HDAC11 on the Sox2 promoter through the Gli1 binding site (G). Myc was used as a control (H). The ChIP qPCR was analyzed using two-way ANOVA test; *p < 0.05, **p < 0.01. Full size image

Depletion of HDAC11 ablates downstream targets and abrogates self-renewal of stem-like cells

Since the earlier results indicated that HDAC11 could induce Sox2-luc promoter activity, we attempted to investigate the role of HDAC11 in expression of Sox2 and its potential downstream targets. H1650 cells were stably transduced with two different IPTG-inducible HDAC11 shRNA. Treatment of the cells with 500 μM IPTG for 5–7 days efficiently depleted HDAC11; there was a concomitant reduction in Sox2 protein levels, as seen by western blotting (Fig. 4A). We also observed a decrease in the expression of YAP1 and Gli1 proteins upon depletion of HDAC11 (Fig. 4A). This was further confirmed by quantitation of the band intensities using ImageJ analysis software (Fig. 4B). Since it has been suggested that Sox2 might affect energy metabolism in stem-like cells, we examined if depleting HDAC11 might affect any metabolic genes that were potentially downstream of Sox231,32. A promoter analysis of glycolysis pathway genes like hexokinase (HK2), Pyruvate dehydrogenase kinase1 (PDK1) and 2 (PDK2) presented several Sox2 transcription factor binding sites (data not shown), suggesting that they are potential transcriptional targets of Sox2. We assessed the expression of these metabolic genes in IPTG treated HDAC11 depleted cells; HK2 and PDK2 showed a significant decrease in their protein expression with the depletion of HDAC11 (Fig. 4A,B). At the same time, there was minimal change in expression of PDK1 at the protein level (Fig. 4A,B).

Figure 4 HDAC11 silencing decreases expression of downstream targets and abrogates self-renewal of CSCs. (A) Depletion of HDAC11 using two IPTG inducible shRNA shows a significant decrease in Sox2, YAP1 and Gli1 protein expression as compared to IPTG treated control shRNA. The glycolysis pathway targets HK2 and PDK2 also show reduced protein expression in the absence of HDAC11. No change is observed in PDK1 expression; the full images of the western blots are provided as Supplementary Fig. 4. (B) Quantitation of the western blot band intensities using ImageJ analysis. (C) Sphere formation assay with SP cells from shHDAC11 H1650 cells treated with IPTG show abrogation of self-renewal ability in the absence of HDAC11. (D) Quantitation of sphere formation assay reveal that IPTG treated SP cells from two different clones of HDAC11 shRNA containing H1650 form fewer spheres as compared to IPTG untreated or control shRNA containing cells. (E,F) Real time PCR analysis of H1650 cells with two different HDAC11 shRNA show a decrease in mRNA expression of Sox2, HK2, PDK1, PDK2 with IPTG treatment. No significant change was observed in Oct4 and Nanog expression. The depletion of HDAC11 was confirmed using RT-PCR. Full size image

Since HDAC11 expression was elevated in the SP cells, we examined if depletion of HDAC11 affected the self-renewal of these cells. SP cells isolated from H1650 cell line stably expressing HDAC11 shRNA were treated with 500 μM IPTG in stem cell selective media for 10 days. It was found that the depletion of HDAC11 reduced the self-renewal of CSCs markedly; IPTG treatment had no effect on SP cells isolated from H1650 cell line stably expressing a non-targeting, control shRNA (Fig. 4C). Quantitation of the spheres revealed that the reduction in sphere formation was significant (Fig. 4D).

Given the above result, we examined if the stem cell transcription factors as well as Sox2 target genes were affected by stable depletion of HDAC11. RT-PCR experiments showed a significant decrease in Sox2 mRNA expression (Fig. 4E) with no similar changes in Oct4 and Nanog expression in HDAC11 knock down cells upon treatment with IPTG (Fig. 4E). Similar reduction was also observed in the levels of HK2, PDK1 and PDK2 mRNAs, compared to IPTG treated shControl cells (Fig. 4F). These results suggested that HDAC11 function was necessary for the self-renewal of SP cells and this was probably mediated through the expression of Sox2 and its targets.

Selective HDAC11 inhibitors reduced stem-like properties of CSCs

To further investigate inhibition of HDAC11, we utilized highly selective and potent inhibitors of HDAC11 those were developed by FORMA Therapeutics33,34. We focused on two of these inhibitors FT234 and FT895 (Fig. 5A); the structurally related FT650 was used as an inactive control.

Figure 5 Selective HDAC11 inhibitors ablates stem-like properties of cancer stem cells. (A) Structures of the three HDAC11 inhibitors used in the study. (B) Sphere formation assay with SP from H1650 cells in the presence of 2 μM concentration of HDAC11 inhibitors FT234 and FT895 show a significant reduction in self-renewal abilities of these cells. FT650 did not have an effect on the self-renewal. (C) Quantitation of the sphere assay confirmed the reduction in the number of sphere formed by H1650 SP cells in the presence of various concentrations of HDAC11 inhibitors FT234 and FT895. The inhibitor FT650 did not affect the self-renewal ability (D) H1650 SP cells treated with 5 μM concentration of HDAC11 inhibitors FT234 and FT895 show abrogation of angiogenic tubule-like structure formation when grown on Matrigel in stem cell medium. FT650 had minimal effect on vascular mimicry of H1650 SP cells. (E) RT-PCR analysis of 2.5 μM HDAC11 inhibitor FT234 and FT895 treated SPAdh reveal decrease in Sox2 and metabolic targets HK2 and PDK2 mRNA expression as compared to DMSO control treatment. FT650 had no effect as seen before. Full size image

Treatment of the stem-like SP cells with 2μM of the selective HDAC11 inhibitors FT234 and FT895 led to a significant reduction in self-renewal as compared to the DMSO treated control (Fig. 5B). The structurally similar but inactive compound FT650 had minimal effect on self-renewal (Fig. 5B). Quantitation of the number of spheres formed in the presence of various concentrations of FT234, FT895 and FT650 revealed a dose dependent inhibition upon treatment with FT234 as well as FT895 (Fig. 5C). No such effect was observed with FT650 (Fig. 5C).

It has been shown that CSCs can undergo vascular mimicry where they are able to form vasculogenic networks on matrigel35,36. Our earlier studies had shown that side population isolated from H1650 cells can undergo vascular mimicry efficiently17,18. There was a significant reduction in the formation of vascular networks by SP cells when treated with 5 μM of FT234 or FT895, as compared to DMSO treatment (Control) (Fig. 5D). A treatment of SP cells with 5 μM of FT650 did not have an effect on the vascular mimicry (Fig. 5D). Thus, it appears that these highly selective inhibitors of HDAC11 could significantly reduce self-renewal of stem-like cells and inhibit their trans-differentiation into vascular cells.

To investigate the essential genes and regulatory pathways influenced by HDAC11 inhibitors in stem-like SP cells, we attempted to generate adherent cultures of the SP cells as described in our earlier publications17,37. Such SP-adherent (SPAdh) cells maintained SP phenotype in 80% of the cells up to 8 days in culture17. When these SPAdh cells were treated with HDAC11 selective inhibitors FT234 and FT895, there was a significant decrease in the mRNA of Sox2 as well as its target genes like HK2 and PDK2, as compared to SPAdh cells treated with either DMSO control or the inactive FT650 compound (Fig. 5E). These experiments suggest that inhibition of HDAC11 can lead to a downregulation of Sox2 as well as its metabolic targets in the stem-like cells, abrogating self-renewal as well as the vascular mimicry of SP cells.

HDAC11 inhibitors prevent tumor cell migration and anchorage independent growth

We next examined the effect of HDAC11 inhibitors on overall cell viability by performing MTT assays. Our results show that 5–10 μM of FT234 compound inhibited the growth and viability by 60–80% in both A549 (Fig. 6A) as well as H1650 cells (Fig. 6B) as compared to the DMSO control treated cells. The treatment with negative control FT650 had no effect on the viability of both the cells (Fig. 6A,B). To further understand the efficiency of the inhibitors, we estimated the IC 50 values for cell viability in the presence of the inhibitors in multiple cell lines namely, A549, H1650, AALE (immortalized tracheobronchial cells) and primary lung CAFs (Supplementary Fig. 2). Table 1 showed the IC 50 values for cell viability. The overall IC 50 value in the primary cells (AALE and lung CAFs) was higher as compared to the cancer cells (Table 1). Also, the FT650 didn’t affect the viability of the cells tested (Supplementary Fig. 3 and Table 1). The IC 50 values for the cancer cell lines A549 and H1650 ranged from 4.663–6.594 µM whereas in AALE the values were 10.74 µM (FT894) and 15.11 µM (FT234). The IC 50 values for primary lung CAFs were 21.34 µM (FT894) and 99.82 µM (FT234) (Supplementary Fig. 3 and Table 1). It should be mentioned that off-target effects are a possibility at these high doses tested in the normal cells. It is likely that the more mesenchymal features of the CAFs contribute to the reduced sensitivity to the HDAC11 inhibitors.

Figure 6 HDAC11 inhibitors prevent tumor cell growth, viability and migration. (A,B) Cell viability assays performed on A549 (A) and H1650 (B) cells with various concentrations of FT234 show a significant decrease in viability of both the cell lines at 5 and 10 μM concentrations. The negative control FT650 had no such effect. (C) Treatment of A549 and H1650 cells with 10 μM FT895 for 24 h abrogates the ability of these cells to migrate in a wound healing assay as compared to the DMSO control treated cells. (D) HDAC11 inhibitors FT234 and FT895 at 10 μM concentration prevent angiogenic tubular extensions in HUVEC cells in a FIBA assay. The negative control FT650 had minimal effect on the tubule growth. (E) The anchorage independent growth of A549 cells assessed in a soft agar colony formation assay in the presence of FT234 and FT650 show a significant decrease in the number of colonies formed in FT234 treatment as compared to DMSO Control treatment. The number of colonies in FT650 treatment as negative control was comparable to DMSO treatment. Full size image

Table 1 IC 50 value for cell viability of cells treated with HDAC11 inhibitors. Full size table

Cell migration and invasion are essential for tumor metastasis38,39. To investigate the effect of the HDAC11 inhibitors on cell migration, a wound healing assay was performed on plastic in the presence of the HDAC11 inhibitors. It was found that cells treated with 10 μM FT895 for 24 h migrated at a slower rate as compared to the DMSO control treated A549 and H1650 cells (Fig. 6C). It is also known that migration is an essential feature in the process of neoangiogenesis and vessel development by the endothelial cells during tumor growth40. We performed a 3D fibrin gel bead assay (FIBA assay) to assess the effect of the HDAC11 inhibitors on angiogenic tubule formation. Treatment with the HDAC11 inhibitors FT234 and FT895 prevented the growth of tubular networks formed by the primary HUVEC cells, as compared to cells treated with DMSO control and the inactive compound FT650 (Fig. 6D). RT-PCR experiments showed that the VEGF receptors, Flt-1 and KDR were suppressed by the HDAC11 inhibitors (data not shown), suggesting a possible mechanism by which angiogenic tubule formation is inhibited by these agents.

Additional experiments were conducted to examine the effect of HDAC11 inhibitors on anchorage independent growth, as this feature strongly correlates with increased tumorigenicity of cancer cells41. Ability of A549 cells to form colonies in soft agar was measured using standard protocols, in the presence of FT234 and FT650. Treatment with 5μM or 10μM the of the compounds for 21 days showed that FT234 could eliminate the anchorage independent growth of A549 cells at both 5 and 10 μM concentrations whereas the inactive control FT650 had no effect on the growth, as compared to the control cells (Fig. 6E). These results strongly suggest that inhibiting HDAC11 using these specific inhibitors might be beneficial in combating cancer, due to their ability to reduce self-renewal, vascular mimicry, migration and adherence-independent growth.

HDAC11 inhibitors reduce Sox2 protein and mRNA expression in cells

Since depletion of HDAC11 selectively suppressed Sox2 expression, experiments were conducted to examine if the HDAC11 inhibitors could affect the expression of Sox2 and its target genes in a similar fashion. A western blot analysis showed that treatment of H1650 cells with 5 μM of FT234 for 72 h decreased Sox2 and YAP1 protein expression as compared to the control cells (Fig. 7A). The FT650 compound had no effect on the expression of either Sox2 or YAP1 (Fig. 7A).

Figure 7 HDAC11 inhibitors reduces Sox2 expression and its regulatory pathways in cells. (A) Western blot analysis with lysates of cells treated with 5 μM of FT234 for 48 h reveal a significant decrease in Sox2 protein expression. There was no change in the expression of Sox2 with FT650 treatment. The full images of the western blots are provided as Supplementary Fig. 5. (B,C) RT-PCR analysis of A549 (B) and H1650 (C) treated with 5 and 10 μM for 72 h with FT234 and FT895 show a significant decrease in Sox2 mRNA expression. There was minimal or no change in Oct4 and Nanog expression. The negative control FT650 also showed no change in the mRNA expression of the ES transcription factors. (D,E) RT-PCR analysis of FT234 and FT895 treated A549 (D) and H1650 (E) reduce the expression of Gli1 and SMO with the treatment. Simultaneously, there was an increase in PTCH1 expression with FT234 and FT895 treatment. As observed earlier, there was no change with the FT650 treatment. Full size image

We next assessed if these specific HDAC11 inhibitors could affect the mRNA levels of the ES transcription factors. The treatment of A549 and H1650 cells with FT234 as well as FT895 reduced the mRNA expression of Sox2 transcription factor (Fig. 7B-C); FT650 didn’t have any effect. Also, there was no change in the expression of Oct4 and Nanog mRNA in A549 and H1650 cells treated with FT234 or FT895 (Fig. 7B-C). This observation was similar to that of depleting HDAC11, suggesting that these novel inhibitors of HDAC11 could exert a similar effect as depleting the protein.

Given the effect of HDAC11 depletion of Gli1 protein, we next examined the effect of FT234 and FT895 on the hedgehog pathway components. Expression of Gli1 mRNA was significantly reduced upon treatment with FT234 and FT895 in both A549 and H1650 cells (Fig. 6D,E). A similar decrease was observed in the levels of Smoothened (Smo) mRNA as well, in both A549 and H1650 cells (Fig. 7D,E). Interestingly, a corresponding increase was observed in the expression of Patched1 (PTCH1) mRNA in A549 and H1650 cells upon treatment with these inhibitors (Fig. 7D,E). Patched1 is known to suppress hedgehog signaling by inhibiting Smoothened19,42 and hence it appears that the HDAC11 inhibitors are downregulating the oncogenic components of the hedgehog pathway, while elevating the levels of the growth suppressive PTCH1.

HDAC11 inhibitors reduce the viability of EGFR TKI resistant cells

Development of drug resistance is widespread in NSCLC patients, and resistance can occur against common chemotherapeutic drugs as well as targeted therapies3,7,43,44. EGFR mutations are prevalent in NSCLC and patients harboring these mutations respond robustly to EGFR TKI inhibitors like erlotinib or gefitinib4,45,46. However, they eventually develop resistance to these drugs, resulting in the resurgence of highly metastatic, drug resistant tumors45. It is widely believed that CSCs contribute to the development of drug resistance and cancer recurrence10,13; since HDAC11 inhibitors could inhibit the self-renewal of CSCs, reduced cell viability and adherence-independent growth of NSCLC cells, we next examined if these inhibitors could eliminate cells those were resistant to EGFR inhibitors. We performed MTT assays on parental HCC827 and erlotinib resistant HCC827 (ER) cells. As can be seen in the Fig. 8A,B, 10 μM FT234 could significantly reduce the viability of HCC827 ER cells (58%), comparable to the parental HCC827 cells (67%). The 2 μM erlotinib treatment eliminated the parental HCC827 cells (Fig. 8A) but not the erlotinib resistant HCC827 ER cells (Fig. 8B). We also conducted similar experiments on viability of parental PC9 cells and gefitinib-resistant PC9 (GR) cells. Here too, 10 μM FT234 significantly reduced the viability of both the parental PC9 cells by (72%) (Fig. 8C) and the PC9 GR cells (80%) (Fig. 8D). 2 μM gefitinib treatment reduced the viability of the parental cells, but not the PC9 GR cells (Fig. 8C,D). These results clearly indicated that HDAC11 inhibitor FT234 could efficiently reduce the survival of the TKI sensitive as well as the resistant cells.

Figure 8 HDAC11 inhibitors reduce the viability of chemo-resistant cancer cells as well as chemo-insensitive CSCs. (A,B) Cell viability assays with HCC827 parental (A) and HCC827 ER (B) cells treated with various concentrations of FT234 show decrease in viability in both the parental and resistant cells at 10 μM concentration as compared to DMSO control treatment. FT650 was a negative control and Erlotinib was used as a positive control. (C,D) Cell viability assays with PC9 parental (C) and PC9 GR (D) cells treated with 10 μM of FT234 reveal a decrease in viability as compared to DMSO control. Gefitinib was a positive control and FT650 was used as a negative control here. (E) Sphere formation assay with FT234 treatment in Cisplatin-insensitive (Cisplatin 2°) cells prevent their self-renewal at 5 μM concentration. (F) Quantitation of the sphere formation assay with cisplatin-insensitive (cisplatin 2°) cells show ablation of number of spheres formed by the cisplatin-insensitive (cisplatin 2°) in the presence of FT234 inhibitor. Such significant decrease was not observed with FT650 treatment. Full size image

Platinum compounds are widely used as chemotherapeutic agents to treat advanced NSCLC and cisplatin is especially efficacious in this regard47. However, cisplatin treatment results in acquired resistance to this drug by promoting multi-drug resistance in CSCs48. Given our observations that HDAC11 inhibitors could eliminate the SP cells that were generally drug resistant, experiments were conducted to assess if HDAC11 inhibitors could eliminate stem-like cells that were insensitive to cisplatin-mediated cytotoxicity. The cisplatin-insensitive cells were generated by treating the SP cells with 5 μM of cisplatin (Supplementary Fig. 4; Materials and Methods). As can be seen from the Fig. 7E,F, treatment with 2 μM and 5 μM of FT234 could inhibit the self-renewal of SP cells that were insensitive to prior cisplatin treatment. The cisplatin insensitive spheres showed comparable inhibition of self-renewal (70%) of the SP cells as the FT234 treatment alone (80%) (Fig. 8F). The FT650 compound had no effect on the self-renewal of SP cells with or without cisplatin treatment. Our earlier studies had shown that Sox2 levels are upregulated in drug resistant cells, and it is likely that inhibition of HDAC11 reduces Sox2 expression, conferring sensitivity to the drugs. These results suggest that inhibition of HDAC11 is a useful avenue to eliminate stem-like cells that are insensitive or resistant to standard chemotherapeutic agents.

HDAC11 inhibitors selectively prevent growth of cancer cells in presence of CAFs

Components of the tumor microenvironment like cancer associated fibroblasts, endothelial cells and immune cells can modulate the growth and progression of tumors49. Cancer associated fibroblasts (CAFs) are known to render drug resistance in various tumor types including NSCLC, mainly through the secretion of growth factors and other survival signals49. We therefore examined the efficacy of the HDAC11 inhibitors on reducing the viability of cancer cells in the presence of primary human lung CAFs. Towards this purpose, we labelled A549 or H1650 lung cancer cell lines with CellTracker red dye and the CAFs with CellTracker green dye and co-cultured them on tissue culture plates in the presence or absence of HDAC11 inhibitors. The results showed that the 10 μM of FT234 inhibitor could selectively prevent the growth of the A549 or H1650 cells (labeled red) by day 3 even in the presence of lung CAFs (labeled green), as seen by the reduction in the red cells and the enrichment of green CAFs (Fig. 9A, Supplementary Fig. 4A). The FT650 compound didn’t inhibit the growth of A549 or H1650 cells, and there appeared to be more red cells compared to green CAFs after three days in co-culture (Fig. 9A, Supplementary Fig. 4A). This experiment suggests that FT234 may eliminate cancer cells selectively even when untransformed CAFs are present.

Figure 9 HDAC11 inhibitors prevent growth of cancer cells even in presence of CAFs. (A) Treatment of 2D coculture of A549 (red) cells with primary lung CAFs (Green) with 10 μM of FT234 show that the HDAC11 inhibitor selectively reduces the growth of A549 cells as compared to the Control co-cultures in 48 h. Such an effect was not observed in the negative control FT650 treatment. (B) 3D coculture assay with labeled A549-luc cells (red) and primary lung CAFs (Green) in non-adherent conditions show a decrease in the growth of A549-luc cells in the presence of 10 μM FT234 in 4 days as compared to control co-cultures. The FT650 did not affect the growth of A549 or CAF cells. (C) Quantitation of the luciferase activity as a measure of the viability and growth of the A549-luc cells in the presence of the HDAC11 inhibitors reveal that FT234 reduces the luciferase activity of these cells as compared to the DMSO control (p = 0.0164) or the structurally similar negative control FT650 (p = 0.0261). Full size image

Given that tumors grow in three dimensions, we examined if HDAC11 inhibitors could eliminate cancer cells selectively in 3D cultures as well. The 3D co-culture assays were carried out in both A549 and H1650 cells. The A549 cells used were stably transfected with a luciferase gene (A549-luc) to enable their quantitation. Both A549-luc and H1650 were labeled with CellTracker red dye and primary lung CAFs were labeled with CellTracker green dye and co-cultured in 1:1 ratio in the presence or absence of the HDAC11 inhibitors in non-adherent condition. The results showed that the 10 μM of FT234 could inhibit the growth and viability of the A549-luc as well as H1650 cells even in the presence of the CAFs in the 3D co-cultures (Fig. 9B, Supplementary Fig. 4B). FT650 did not inhibit the viability of either the cancer cells or the lung CAFs. The inhibition of the growth and viability by HDAC11 inhibitors was monitored in A549-luc cells by measuring luciferase activity using luciferin as a substrate. As seen in Fig. 9C, FT234 treatment significantly reduced luciferase activity in A549 cells as compared to the FT650 or the DMSO control treatment, indicating that HDAC11 inhibitors can efficiently prevent the growth of A549 cells even when CAFs are present. The results confirm the efficacy of the HDAC11 inhibitors against drug insensitive CSCs that may arise during the chemotherapeutic treatments, indicating that HDAC11 inhibitors might be potentially useful in treating NSCLC.