ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling

Abstract

Objective: Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance.

Methods: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators.

Results: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells.

Conclusion: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

Figure 1. ALDH1A1 status correlates with platinum resistance in ovarian cancer cells.

Isogenic cell lines (A2780- platinum sensitive & A2780/CP70- platinum resistant) were evaluated for platinum response using drug sensitivity assay as described in M&M section (A). To assess the percent of ALDH+ cells, in A2780 and A2780/CP70 cells ALDEFLUOR assay was performed by using BODIPY-aminoacetaldehyde (BAAA) as substrate for ALDH enzyme, after 40 minutes incubation at 37°C flow cytometry analysis was performed (B) and western blot data shows representing levels of ALDH1A1 isozyme in these cells (C).

Figure 2. ALDH1A1 status correlates with progression free survival in ovarian cancer patients.

Ascites from 15 consecutive patients with primary advanced stage III/IV ovarian cancer and 2 from benign (Miegs syndrome) was obtained and analyzed for percentage of ALDH+ cells through ALDEFLUOR assay. The histogram in inset shows the percent of ALDH+ cells in benign and malignant ascites (A). The clinicopathologic information from the ovarian cancer patients were correlated with the percent of ALDH+ cells in their ascites and evaluated progression free survival with ALDH^HIGH (>15% ALDH) to ALDH^LOW (<15% ALDH) patients (3 vs. 9 months, respectively; p<0.01) (B).

Figure 3. ALDH+ phenotypes demonstrate cancer stem cell properties.

A2780/CP70 cells were sorted into ALDH− and ALDH+ phenotypes and evaluated for their abilities for invasion using Matrigel invasion chambers (A), quantitative data as represented (B) and colony formation potential in the presence and absence of carboplatin (20 µM) were assessed using soft agar colony formation assays (C). In order to determine possible mechanism for cancer stem cell characteristics in ALDH+ cells, we evaluated multiple markers of “stemness”. A2780/CP70 cells were sorted into ALDH− and ALDH+ phenotypes, total RNA was isolated, cDNA was prepared and real-time quantitative RT-PCR was performed (D). **indicates statistical significance (p<0.01).

Figure 4. ALDH1A1 down regulation differentially affects on cancer stem cell properties.

Lentiviral vectors expressing ALDH1A1 specific shRNAs or nontargeting shRNAs were transfected into A2780/CP70 cells and its effect on stem-like cell properties were evaluated for invasive potential using Matrigel invasion chambers (A), quantitative data as represented (B), clonogenic potential using soft agar colony formation (C, and carboplatin dependent growth inhibition by CellTiter-Glo Luminescent Cell Viability Assay (D). Statistical significance was evaluated using student’s t-test.

Figure 5. ALDH1A1 downregulation leads to lower KLF4 & p21 expression with altered cell-cycle profile.

ALDH1A1 proficient and deficient A2780/CP70 cells were evaluated for expression of stem-like cell marker KLF4 and cell cycle checkpoint protein p21 by RT-PCR and Western blot analysis (A). Cell cycle distributions of cells were analyzed after fixing the cells in 70% methanol and propidium iodide staining followed by flow cytometry (B). Apoptotic cells were detected after staining the cells with APC-Annexin V and 7-AAD according to the manufacturer’s instructions and analyzed by flow cytometry (C).

Figure 6. ALDH1A1 status alters cell cycle checkpoints and DNA repair networks.

ALDH1A1 proficient (control) and deficient (shALDH1A1) A2780/CP70 cells were assessed for their abilities to repair carboplatin induced single strand breaks by looking at time dependent induction of PARP-1 protein levels by western blots (A), densitometry of blots by Image J (B) and total PARP activity was assayed by measuring PAR levels (C). To assess the ALDH1A1 dependent expression DDR and repair proteins, whole cell lysates were normalized for total proteins and western blot analysis were performed using antibodies as represented (D).