Specifically targeting ERK1 or ERK2 kills Melanoma cells

The human melanoma cell line A375 was purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM (Lonza, Walkersville, MD, USA) plus 10% FCS (Gemini Bio-Products, Woodland, CA) in a humidified incubator (37°C, 5% CO₂). Annexin-FITC was purchased from Biovision Research Products (Mountain View, CA, USA), and tetra methyl rhodamine ethyl ester (TMRE) was purchased from Invitrogen Molecular Probes (Eugene, OR, USA). Pan-caspase inhibitor ZVAD was purchased from BD Biosciences (San Jose, CA, USA). Propidium iodide (PI) was purchased from Sigma Chemical Co (St Louis, MO, USA). PD0325901 and PLX4032 were purchased from Biovision Research Products (Mountain View, CA, USA) and Selleck Chemicals (Houston, TX, USA), respectively. Abs against ERK1, ERK2, pERK1, pERK2, MEK, pMEK, p-Bad, Bak, Bim, PUMA were purchased from Cell Signaling Technology (Beverly, MA, USA); whereas Bcl-XL, Mcl-1, Bad, PARP, caspase 3, Raf-1, Raf-B and GAPDH were purchased from Santa Cruz (Santa Cruz, CA). Ab against Bcl-2 was from DAKO (Glostrup, Denmark), ab against actin from Chemicon Int. (Billerica, MA, USA); Ab against Bax from Calbiochem (San Diego, CA, USA), and against XIAP and activated Bax from BD Transduction Lab (Franklin Lakes, NJ, USA). Primary Abs incubated overnight at 4°C, and secondary Abs were incubated at room temperature for 1 hr.

Mission TCR shRNAs targeting human ERK1 (NM_002746) and ERK2 (NM_138957) were purchased from Sigma Chemical Co. pLKO.1 Scramble control shRNA plasmid, psPAX2 packaging plasmid and pMD2.G envelope plasmid were provided by Addgene. To make lentiviral particles, HEK-293 T cells were plated into 10 cm plates, 2 × 10⁶ cell/plate, with 8 ml of DMEM plus 10% FBS and no antibiotics. On the next day, for each plate 3 ug of pLKO.1 shRNA plasmid together with 2.25 ug of psPAX2 and 0.75 ug of pMD2.G plasmid were transfected with FuGen 6 reagents (Roche, New Jersey) according to the manufacture's instruction. The transfection reagent was removed by replacing the medium with fresh DMEM containing FBS and penicillin/streptomycin on the following day. The cells were incubated at 37°C, 5% CO₂ for 24 hr for another 2 days. Supernatants from 24 hr and 48 hr incubations were harvested and combined followed by centrifugation to remove cell debris and stored at -80°C.

A375 melanoma cells were plated onto 6 well plates at 3 × 10⁵ cells/well and incubated at 37°C, 5% CO₂ overnight. Cells were washed 1x with PBS and 1 ml of lentiviral supernatants containing shRNA for either ERK1 or ERK2 or scramble control was added in each well. For ERK1 and ERK2 double knockdown, both supernatants (1 ml of each) were added into one well. All viral supernatants were added with hexadimethrine bromide (Sigma Chemical Co.), final concentration 8 μg/ml) before use. After 4-6 hr incubation, supernatants were changed with fresh medium and cells were incubated for another 1 or 2 days before being split for experiments.

Cell death was measured by flow cytometry after staining cells with Annexin-V-FITC and I mg/ml of PI. To investigate DNA degradation, one of the hallmarks of apoptosis, cells were fixed with 70% ethanol and PI stained in the presence of RNAse (10 μg/ml). The relative percentage of cells with hypo-diploid DNA content (sub-G₀) was determined by FACS analysis and use of Excel software.

For anchorage independent colony formation assay, A375 cells were transduced with shRNAs for 2 days and then suspended in DMEM with 0.5% agarose solution, and plated onto solidified 1% agarose in 6 well plate at a density of 2500 cells per well in triplicate. A375 cells were maintained in culture by feeding with 0.5 ml fresh DMEM plus 10% FBS medium twice a week, for a total 3 weeks. Two independent assays were carried out and the number of colonies were counted after staining with 0.1% crystal violet solution at the end of each experiment. For anchorage dependent colony assay, A375 cells infected with shRNAs for 2 days were seeded into 6 well plates at a density of 2000 cells per well in triplicate. The cells were cultured with complete DMEM for another 9 days and colony number counted after being stained with 0.1% crystal violet solution.

Assessment of mitochondrial membrane potential was determined by addition of 100 nM of TMRE dye that accumulates in mitochondria of living cells [29]. Reduction in TMRE retention is indicative of loss of mitochondrial membrane potential.

A375 cells were fixed with 2.5% paraformaldehyde (10 min, room temperature), washed and incubated with a primary ab detecting the activated configuration of Bax (BD Pharmingen Inc, San Diego, CA, USA) in FACS buffer with 0.3% saponin as previously described [30]. The percentage of melanoma cells with activated Bax was measured by fluorescence intensity greater than control ab levels.

Western blot analysis was performed as previously described [31]. Briefly, cells were harvested by scraping and lysed with M-Per mammalian protein extraction reagent (Thermo Scientific, Rockville, IL, USA) supplemented with protease inhibitor cocktail (Roche Diagnostics GmbH, Germany) and phosphatase inhibitor cocktail set II, (Calbiochem, Los Angeles, CA, USA) followed by shaking and centrifugation at 4°C. Supernatants were collected as whole cell extracts and protein concentrations were measured using Bradford reagents (Bio-Rad laboratories, Hercules, CA, USA). 30 ug of proteins were resolved by SDS- PAGE and transferred to PVDF membrane followed by 1 hr blocking with buffer supplied by LI-COR Biosciences (Lincoln, NE, USA). Blots were probed with primary Abs overnight at 4°C, washed and incubated with corresponding fluorescence-labeled secondary Ab for 1 hr at room temperature in dark. Protein levels were visualized with LI-COR Infrared Imaging System.

Total RNA was extracted from A375 cells using Trizol (Invitrogen Life Technologies, Inc.). One microgram of RNA was reverse transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). The following specific primer pairs were used: BRAF: 5'-CTC GAG TGA TTG GGA GAT TCC TGC-3', (forward), 5'-CTG CTG AGG TGT AGG TGC TGT CAC-3' (reverse); 18sRNA: 5'-GGC GCC CCC TCG ATG CTC TTA G-3', (forward), 5'-GCT CGG GCC TGC TTT GAA CAC TCT-3', (reverse). PCR reaction was performed by adding 25 μl of 2X SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA), 19 μl DEPC treated H₂O, 2 μl of each primer, 2 μl of diluted cDNA template. DNA amplification was completed in ABI prism model 7300 thermal cycler. All reactions were run in triplicate and two independent assays were performed. The comparative expression level was determined by applying the calculation of 2^{(Δ Ct B-raf-ΔCt 18s).}

All statistical analyses were performed using the unpaired, two sided Student's t-test, and results considered significant when P values were less than 0.05.

In A375 cells, constitutive levels for ERK2 are slightly greater than ERK1, and the pERK2 is also slightly more abundant compared to pERK1 (Figure 1A). As there are currently no chemical inhibitors to selectively block ERK1 versus ERK2 activity [32], lentiviral preparations containing specific shRNAs were used targeting ERK1 or ERK2. Addition of ERK1 shRNA significantly reduced protein levels of ERK1 and pERK1 compared to scrambled control shRNA treatment, as observed beginning on day 2, with greater reduction on day 4 and near complete absence on day 6. Interestingly, reduction in ERK1 levels was accompanied by minimal changes in the protein level of ERK2 and pERK2 (Figure 1A, left side panel). Similarly, addition of ERK2 shRNA significantly reduced protein levels of ERK2 and pERK2 compared to scrambled control shRNA treatment with similar kinetics as observed for ERK1 shRNA, with minimal changes in Erk1 and pErk1 protein levels by the ERK2 shRNA. Quantitative analysis of a representative blot on day 4 is presented in Figure 1A-right side panel. Thus, despite both ERK1 and ERK2 are MEK substrates; significant reductions in one isoform did not trigger increased phosphorylation of the other isoform in A375 cells.

Treatment with shRNAs silencing ERK1 and/or ERK2 did not trigger detectable killing of A375 cells 2 days after infection (although G₁ growth arrest was noted, data not shown), but progressively increased killing was observed 4 days (p < 0.0001 for ERK1 and ERK1 plus ERK2 shRNAs; and p < 0.002 for ERK2 shRNA) and 6 days (p < 0.002 for ERK1 shRNA and p < 0.001 for ERK2 and ERK1 plus ERK2 shRNAs) after infection (Figure 1B). Note by combining ERK1 plus ERK2 shRNA infections, no significant additive or synergistic increased killing was observed. To determine the mode of cell death cells with sub G₀ DNA content were also analyzed and found to be increased (p < 0.03 for ERK1 or ERK2 shRNAs at 4 days), and p < 0.001 for ERK1 plus ERK2 shRNA at 6 days pointing to a role for apoptosis in the response to the ERK shRNAs (Figure 1C).

Next, the biological effect of a sustained reduction in pERK1 and/or pERK2 on anchorage independent colony formation in soft agar (Figure 2A) was determined (21 days). There was significant reduction in melanoma cell colony forming potential followed treatment with ERK1 shRNA or ERK2 shRNA; with virtually no colonies forming following combined ERK1 plus ERK2 shRNAs (all values p < 0.001) as assessed by counting the number of clones per 10X magnification light microscopy (Figure 2A lower panel).

To assess potential of anchorage dependent colony formation, clones of A375 melanoma cells developing resistance to ERK1 and/or ERK2 shRNA treatment were determined by visual inspection and manual counting of the numbers of clones developing after 9 days (Figure 2B). While numerous and easily visible clones were apparent when cells were treated with SC shRNA, treatment with the ERK shRNAs did not generate any resistant clones when used in combination, and only rare clones could be identified with single treatment, being greater for ERK2 shRNA than ERK1 shRNA as assessed by counting the number of clones per well (Figure 2B lower panel; all reductions p < 0.001).

Western blot analysis revealed silencing either ERK1 and/or ERK2 triggered cleavage (activation) of caspase 8 after 4 days and 6 days, with cleaved caspase 3 detected after 6 days of treatment (Figure 3A). PARP was cleaved more prominently by ERK2 shRNA compared to ERK1 shRNA (similar to caspase 8) on day 4, and by day 6 ERK1 and/or ERK2 shRNAs triggered near complete cleavage of PARP. The role of caspases in killing of A375 cells was studied using the pan-caspase inhibitor, ZVAD (Figure 3B); which revealed significant blocking of day 4 cell death by ERK1 shRNA (p < 0.01; p < 0.002 for ERK2 shRNA and p < 0.02 for ERK1 plus ERK2 shRNAs).

To assess the integrity of the outer mitochondrial membrane potential before and after 2, 4 and 6 days of treatment, A375 cells were labeled with a TMRE dye (Figure 4A), and reduction in the intensity of staining indicating altered membrane potential was progressively increased over the time course for ERK1, ERK2 and ERK1 plus ERK2 shRNAs in A375 cells. Further analysis using abs detecting the activated forms for Bax in permeabilized cells with FACS (Figure 4A-lowest panel set), revealed all shRNA treatments triggered increased levels of activated Bax within the melanoma cells.

Since cell death is regulated by relative levels of various pro-apoptotic and anti-apoptotic proteins (Figure 4B), cell extracts were prepared before and 4 days after various treatments and western blot analysis included a panel of anti-apoptotic proteins (Figure 4B upper panel), and pro-apoptotic proteins (Figure 4B lower panel). Constitutive levels for proteins associated with cell survival were detected for Bcl-X_L, Mcl-1, Bcl-2, and XIAP (Figure 4B upper panel). Treatment with ERK1 and/or ERK2 shRNA did not lower these levels. There was slight increase in XIAP by knocking down ERK1 but not ERK2, which did not seem to protect cells from the killing by targeting ERK2. As regards the pro-apoptotic protein levels (Figure 4B lower panels), knockdown of ERK1 and/or ERK2 increased levels of Bak, Bax, Bad, Bim with variable changes in Noxa and PUMA levels, accompanied by decreased p-Bad levels in the A375 cells. Both Noxa and PUMA appeared to be dependent on ERK1, consistent with recent report [33]. Overall, targeting ERKs appeared to more significantly influence pro-apoptotic protein levels over pro-survival protein levels.

Treatment with the MEK inhibitor, PD0325901 reduced ERK1 levels at 4 hr and 8 hr, but eliminated pERK1 and pERK2 levels between 2 hr and 48 hr (Figure 5A), with sustained reduction after 4 days (Figure 5B). Treatment with mutant BRAF specific inhibitor, PLX4032 slightly influenced ERK1 and ERK2 levels, but eliminated pERK1 and pERK2 levels up to 24 hr; but this was not sustained as both phosphorylated isoforms were detectable after 48 hr and 4 days (Figure 5A, B, respectively). Induction of A375 cell killing was rapid (Figure 5C) as detected at the 2 day assay point, (approximately 20% of the cell population; p < 0.01 for both drugs), with slight increases on day 4 (p < 0.001 for both drugs) or day 6 (p < 0.001 for PD0325901 compound and p < 0.01 for PLX4032).

PD0325901 treatment triggered slight reductions in Bcl-X_L, Mcl-1, Bcl-2 after 4 days of treatment, but not XIAP; whereas PLX4032 triggered greater reductions all of these pro-survival protein levels, but not XIAP (Figure 5D). Pro-apoptotic protein levels were either relatively unchanged or diminished by treatment with these two drugs, which may reflect the overall relatively modest levels of killing achieved during the six-day period of treatment with PD032509 or PLX4032.

To explore the molecular basis for lack of emergence of treatment resistance clones when using ERK1 and/or ERK2 shRNAs, relative levels of upstream kinases were determined 4 days after treatment (Figure 6A-left side panel). Compared to levels observed using SC shRNA, the levels of BRAF, CRAF, phospho-MEK, but not total MEK were significantly reduced by either ERK1 shRNA or ERK2 shRNA, and for BRAF became even lower with ERK1 and ERK2 shRNA combined. Given the reports of treatment resistance of melanoma cells to PLX4032, we compared and contrasted the same upstream kinase levels after 4 days of treatment with either PLX4032 or PD0325901 (Figure 6A-right side panel). Compared to untreated A375 cells, continuous exposure to PD0325901 lead to increased levels of BRAF, which was also modified so as to reduce its electrophoretic motility, accompanied by near complete loss of CRAF and increased phospho-MEK and slightly reduced total MEK levels. By contrast, PLX4032 treatment did not reduce BRAF levels, although both CRAF and phospho-MEK levels were dramatically reduced. Since BRAF levels appeared to discriminate between the shRNA treatment that suppressed resistant clone formation and PLX4032 that promoted resistant clone formation, relative mRNA levels were determined using RT-PCR (Figure 6B). Compared to untreated cells, PD0325901 increased BRAF mRNA levels, but none to relatively minor changes in BRAF mRNA levels were observed for all other treatments (PLX4032, ERK1 and/or ERK2 shRNAs), indicating changes involving non-transcriptional regulatory elements.

As noted in Figure 5A, treatment of A375 cells with PLX4032 led to a rebound in pERK levels after 48 hrs, and to determine if this rebound level of pERK contributed to melanoma treatment resistance associated with PLX4032 treatment, we first knocked down pERK levels with the ERK shRNAs (which generate sustained and selectively knockdown of each pERK isoform (Figure 1A), and then added PLX4032. Compared to the killing of A375 cells accomplished by either use of ERK shRNAs or PLX4032 alone (Figure 7), greater killing was triggered by PLX4032 in the co-presence of ERK1 shRNA, ERK2 shRNA or ERK1 plus ERK2 shRNA (p < 0.001 for all combinations compared to single treatment values).