Targeting aldehyde dehydrogenase cancer stem cells in ovarian cancer

Abstract

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P < 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P < 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer.

Figure 1. Comparison of whole genome expression profiling between SKOV3TRip2 and SKOV3ip1 cell lines

Total RNA from the SKOV3TRip2 and SKOV3ip1 cell lines were subjected to whole genome expression profiling using the Illumina platform. The genes with a greater than 10-fold increase (FC-fold change) in SKOV3TRip2 are shown in red, those with a greater than 10-fold increase in SKOV3ip1 are shown in green.

Figure 2. ALDH1A1 expression in ovarian cancer cell lines

A) As measured with the MTT viability assay, the SKOV3TRip2 ovarian cancer cell line has a docetaxel IC50 approximately 3,000-fold higher than its parental SKOV3ip1 cell line. B) Expression of ALDH1A1 by Western blot in four pairs of chemosensitive and chemoresistant ovarian cancer cell lines, and the nontransformed HIO-180 normal ovarian surface epithelium line. In all cases except HeyA8/HeyA8MDR, in which both lines had minimal expression, the chemoresistant line had increased ALDH1A1 expression. C) As measured by the ALDEFLUOR assay, the A2780cp20 (cisplatin resistant) and SKOV3TRip2 (taxane resistant) also contained a higher percentage of cells with functional ALDH1A1. D) This was confirmed by immunohistochemistry (IHC) for ALDH1A1 on these cell lines in vitro, where individual cells appeared either negative or strongly positive, demonstrating heterogeneity of ALDH1A1 expression in the cell line population. A low-power (4x) view gives an appreciation for the distinct colonies of ALDH1A1-positive cells, while examination at high power (10x) shows the definitive ALDH1A1-positive or negative nature of individual A2780cp20 and SKOV3TRip2 cells, but an absence of ALDH1A1 in parental A2780ip2 and SKOV3ip1 lines. E) This heterogeneity is also present in tumor xenografts, as seen with IHC for ALDH1A1 in SKOV3TRip2 tumors grown in mice (intraperitoneal location is confirmed by presence of normal pancreatic tissue on the right side of the slide) F) ALDH1A1 expression is not limited to hypoxic cells, as demonstrated in xenografts collected from mice given the hypoxyprobe reagent and subjected to co-IHC for ALDH1A1 (in blue) and the hypoxyprobe byproduct (in brown). Scale bars represent 50µm in 10x views, 100µm in 4x views and (E, F).

Figure 3. ALDH1A1 expression in ovarian cancer patients

ALDH1A1 was assessed by IHC in 65 high-grade stage III–IV papillary serous ovarian cancer patients. A) Several expression patterns were seen, including absent, spotty (e.g. “Low ALDH”), and diffuse (“High ALDH”) staining. Consistent with staining in cell lines, both strongly positive and negative populations were noted. B) To confirm the “spotty” ALDH1A1 pattern was not identifying infiltrating macrophages, co-IHC on frozen tissue for ALDH1A1 (brown) and CD68 (a pan-macrophage marker, blue) was performed. C) Patients were stratified into less than 1%, 1–20% and greater than 20% ALDH1A1 expression, and progression-free and overall survival plotted by the Kaplan-Meier method and tested for statistical significance with the log rank test. There was a significantly shorter progression-free survival in patients with increasing ALDH1A1 expression. D) Mice with established primary subcutaneous xenografts were treated with vehicle or cisplatin for 5 weeks. E) Tumors from these mice were harvested and subjected to IHC for ALDH1A1. Tumors treated with cisplatin demonstrated a significant increase in the number of ALDH1A1-positive tumors cells. Magnification at low and high power are shown; scale bars represent 50µm in (A, B), and high power (E), 100µm in low power (E).

Figure 4. ALDH1A1 populations in A2780cp20 xenografts

Intraperitoneal tumors that developed after injection of ALDEFLUOR–positive or negative A2780cp20 cells were assessed for ALDH1A1 composition. A) Tumors that formed after injection of purely ALDEFLUOR-positive cells demonstrated both ALDEFLUOR-positive and negative populations, and recapitulated the TIC phenotype of having a small (2.4–6.1%) percentage of ALDEFLUOR-positive cells. B) Interestingly, tumors that formed after injection of purely ALDEFLUOR-negative cells contained only ALDEFLUOR-negative cells, showing an absence of capacity for differentiation, at least in terms of ALDEFLUOR positivity. C–D) This expression discrepancy was also noted on IHC for ALDH1A1 from these samples. Scale bars represent 100µm. E) Similarly, in vitro, SKOV3TRip2 ALDEFLUOR-positive cells give rise to both ALDEFLUOR-positive and ALDEFLUOR-negative cells, re-establishing baseline levels at 48 hours, whereas (F) ALDEFLUOR-negative cells cannot give rise to ALDEFLUOR-positive cells.

Figure 5. Efficacy of ALDH1A1 downregulation with siRNA in vitro and in vivo

A) Identification of siRNA constructs that decrease ALDH1A1 expression were confirmed with Western blot, and B) by flow cytometry using the ALDEFLUOR assay in the SKOV3TRip2 cell line. C) Downregulation of ALDH1A1 with siRNA 48hrs prior to treatment of SKOV3TRip2 cells with increasing doses of docetaxel demonstrated a sensitization effect, decreasing the IC50 from 178nM to 82nM. siRNA alone also showed an effect, with decreased viability by 49%. D) In the A2780cp20 cell line, downregulation of ALDH1A1 alone had minimal effect, but sensitized cells to cisplatin, decreasing the IC50 from 5.1µM to 2.0µM. E) Cell cycle analysis shows that ALDH1A1 downregulation induces accumulation of cells in S and G2 phases in SKOV3TRip2, with little effect on A2780cp20. F) In vivo, mice injected intraperitoneally with SKOV3TRip2 cells were treated with ALDH1A1-siRNA incorporated in DOPC nanoparticles, docetaxel/control siRNA in DOPC, or the combination, and compared to mice treated with control siRNA in DOPC. Mice treated with either single agent had minimal effect, but the combination showed a significant reduction compared to treatment with control siRNA (94% reduction in tumor growth, p<0.001) or either single agent (90–91% reduction, p<0.005). G) Similarly, mice injected with A2780cp20 cells showed a minimal non-significant reduction in growth with cisplatin or ALDH1A1-siRNA in DOPC, but combination therapy was statistically superior to either single agent (65–73% reduction, p<0.04), and control siRNA (85% reduction, p=0.048). Mean tumor weight and individual tumor sizes are presented.