Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.


Western blotting
Melanoma cells were treated either with 0.5-1mM phenformin or vehicle for the indicated timepoints. Cells were lysed in Ripa buffer. Lysates were then centrifuged at 13,000 rpm at 4°C for 15 minutes to remove any cell debris. Protein concentrations were determined using Bradford assay (Biorad Laboratories Inc., Hercules CA) according to the manufacturer's protocol. Proteins were then boiled for 5 min, separated by SDS-PAGE with 4-20% gradient gels (Bio-Rad Laboratories inc., Hercules, CA) and transferred to nitrocellulose blotting membranes (Amersham Biosciences, Little Chalfont Buckinghamshire, UK). After an incubation in blocking buffer (5% Non-fat dry milk in phosphate buffered saline (PBS/0.1% Tween), the membranes were incubated with primary antibodies at 4°C. For western blotting analysis, the following antibodies were used: anti-AMPK (Cell Signalling, Hitchen, UK), anti-ALDH1A3 (Abgent, San Diego, CA), anti-MITF (Abcam, Cambridge, UK), anti-Flag (Sigma-Aldrich, St. Louis, MO), anti-SOX-2 (Cell Signalling). β-Actin (Sigma-Aldrich, St Louis, MO) served as a loading control. Bound antibody was detected using anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody and chemiluminescence (ECL Plus Kit, Amersham Biosciences, Little Chalfont Buckinghamshire, UK).

Cell viability assays
Cell viability in 2D-cell culture models was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) and trypan blue (TB) dye exclusion assays, as previously reported. For MTT assay, melanoma cells were seeded into each well of 96-well plates (5,000 cell/well) and treated the next day with vehicle control or biguanides (metformin 0.1, 1, 10mM; phenformin 0.1, 0.5, 1mM) for given incubation time. Viable cells were detected by incubating with MTT (Sigma-Aldrich, St. Louis, MO) solution at 37°C for 4 h. The formazan dye produced by viable cells was solubilized with DMSO and measured by a multiwell scanning spectrophotometer at 540 nm. Results were expressed as mean±SD (n =3). Student's t-test was performed for comparison of the means. TB assay was used to count viable melanoma cells both in monolayer cell culture experiments and when melanoma cells were allowed to form spheroids. For monolayer cell cultures, cells were trypsinized at each timepoint and mixed 1:1 with trypan blue in order to count viable cells. Results were expressed as mean±SD (n =3). For 3D models, spheres were harvested at day 10 or 14, mechanically disaggregated and the number of viable cells was counted by TB dye exclusion using a hemocytometer. Results were reported as the number or % of viable/dead cells/sphere at the indicated timepoint.

Aldefluor assay specifics
After mechanical disaggregation of spheroids, single-cell suspensions were suspended in Aldefluor assay buffer containing BODIPY-aminoacetaldehyde and incubated at 37°C for 30 minutes, as previously shown [4]. A small amount of the same cell suspension was incubated with Aldefluor buffer containing 50 mM diethylaminobenzaldehyde, an ALDH inhibitor. 7-AAD (7-Amino-actinomycin D) was used to exclude dead cells. Cell sorting and ALDH analysis were performed using a FACS-ARIA (Becton Dickinson, Franklin Lakes, NJ) and a FACS-CANTO II (Becton Dickinson), respectively. The results were analyzed using fluorescence-activated cell sorting (FACS) Diva software (Becton Dickinson). The gating strategy included the ALDHhigh gate being set at least one log apart from the ALDH low one. We set a cutoff of 20% ALDH high/low cells in order to better separate the two populations. The purity of sorted populations was analyzed and had to be greater than 95% in order to proceed with the experiments. Sorted cells were either directly lysed for expression analyses or re-seeded to perform proliferation assays.

3D spheroid invasion assay
In method I cells were cultured at 80% confluence, then harvested, counted and resuspended in spheroid formation matrix. This mixture was comprised of 5 µL spheroid formation ECM and 15 µL of medium with FBS and penicillin/streptomycin. Fifty microliters of cell suspension were added per well to the 3D culture qualified 96-well spheroid formation plate and centrifuged at 200 g for 3 min at room temperature and then incubated at 37°C for 72 h to promote spheroid formation. Fifty microliters of the invasion matrix was added to each well in 3D culture 96-well spheroid formation plates. The spheroid formation plate was centrifuged at 300 g at 4°C for 5 min, then transferred to the incubator at 37°C for one hour to promote gel formation. After one hour, 100 µL of complete medium containing vehicle or the indicated doses of metformin and phenformin was added to each well. The spheroid formation plate was incubated at 37°C for 1 to 5 days, and spheroids were photographed in each well every day. Using method II, we analyzed the ability of melanoma cells resistant to metformin or phenformin to preserve invasive capacity. We first generated spheroids structures as described above, then treated them with metformin or phenformin in complete medium for 72h. After pre-treatment, fifty microliters of the invasion matrix was added to each well in the 3D culture-plate. After centrifugation and gel formation, we added complete medium (without biguanides) to each well for 1 to 5 days. For both methods, a set of spheroids seeded without invasion matrix (no-matrix) was prepared in order to calculate spheroids invasion area, which is the quantification of spindle-like projections of the cells in spheroids in presence of invasion matrix. This number is calculated by subtracting the invasion area of no-matrixspheroids to that of spheroids seeded with invasion matrix. In order to measure the viability of cells forming spheroids at the chosen timepoints, we enzymatically digested invasion matrix with 200U/ml dispase for 30 minutes and counted the number of viable cells/sphere by trypan blue cell counting. Results are expressed as the area of invasion over viable cells.

Apoptosis analysis
Apoptosis in SK-MEL-28, A375 and BTC#2 cells was assessed using Annexin V Apoptosis detection kit (BD BD Biosciences Pharmigen, San Diego, CA). Briefly, cells were washed twice with cold PBS and resuspended with 1X Binding Buffer at concentration of 1 X 10 6 cells/ ml. 100 microliters of the solution (1 X 10 5 cells) were incubated with 5 microliters of PE Annexin V and 5 microliters of 7-AAD. Then, cells were gently vortexed and incubated for 15 minutes at room temperature in the dark. 400 microliters of 1X Binding Buffer were added and fluorescence was measured by flow cytometry (Becton Dickinson) and analyzed by FACS DIVA software. Apototic cell death was determined by counting the cells that stained positive for Annexin V. Apotosis was also confirmed by staining the same cells with an hypotonic solution containing 50micrograms/ml propidium iodide, 0.1% sodium citrate and 0.5% tryton X-100. After 15 minutes at 4°C in the dark cells were analysed by flow cytometry. Apoptosis was detected by evaluating the reduced fluorescence of the DNA-binding dye PI in the apoptotic nuclei.