LIDAD DE ESPUMÍGENO

PP2A dephosphorylates a myriad of proteins [279]. In order to understand the signalling effects of PP2A inhibition, alone and in combination with lapatinib, HCC1954-L cells treated with 500 nM lapatinib, 3 nM okadaic acid, and lapatinib plus okadaic acid for 18 h were examined by reverse phase protein array (RPPA) analysis. RPPA experiment was carried out by Dr. Mattia Cremona. HCC1954-L cells were chosen for this experiment as they showed a greater combination effect of okadaic plus lapatinib than the SKBR3-L cell line.

Lapatinib treatment significantly altered the total or phosphorylated levels of 10 proteins (Table 3-2). As lapatinib is an EGFR/HER2 inhibitor, it is unsurprising HER family member levels and activity was altered with lapatinib treatment. Phosphorylated HER2 (Y1248) and HER3 (Y1289) were both significantly decreased

(p = 0.016 and 0.047, respectively). Total protein levels of EGFR were also decreased compared to DMSO-treated cells (p value = 0.009). Downstream of HER2, Akt and Akt2 levels were significantly increased. Phosphorylated Akt (S473 and T308) were slightly reduced but this change was not significant. Interestingly, 18 h lapatinib treated caused increases in levels of the RTK MET. Grb2-associated binding protein 1 (GAB1) phosphorylation was also decreased. GAB1 is an adapter protein that interacts with several RTKs, including EGFR and MET [280, 281]. This may indicate after the 18 h treatment, downstream signalling may be suppressed. Previous work in our lab, showed decreased Akt and ERK 1/2 phosphorylation after 24 h of lapatinib treatment [154].

Eighteen hour 3 nM okadaic acid treatment significantly altered only two phosphorylated protein levels and decreased total levels of one protein, Akt2 (Table 3-3). Okadaic acid caused significant decrease in Src (Y416) levels (p = 0.035) and increased p38 MAPK (T180) (p = 0.039). The RPPA analysis examined 67 total or phosphorylated protein levels. However, PP2A regulates a large amount of proteins outside of the pathways encompassed in this analysis. PP2A inhibition with okadaic acid may be altering proteins outside of the remit of the RPPA platform used.

The combination of 500 nM lapatinib plus 3 nM okadaic acid caused 15 protein expression and activation changes, five of these were common to the lapatinib treatment (Akt, EGFR Y1068, HER2 Y1248, GAB1 Y627, NFκB p65 S536) and one with okadaic acid treatment (Src Y416) (Table 3-4). Combination treatment showed decreased phosphorylation of EGFR (Y1063 and Y1172) (p = 0.003 and 0.0001, respectively), HER2 (Y1248) (p = 0.0025), and HER3 (Y1289) (p = 0.006). Downstream from HER family member signalling, combination treated HCC1954-L cells showed significant decreases in phosphorylation of MAPK (T202/Y204) (p = 0.027), MEK 1/2 (S217) (p = 0.0007), Shc (Y317) (p = 0.04), Src (Y416) (p = 0.016), total and phosphorylated levels of mTOR (S2448) ( p = 0.042 and 0.019, respectively) and total Raf levels (p value = 0.003). Total levels of the pro-angiogenic receptor, VEGFR2, was also decreased (p = 0.042). Akt2 levels were increased with lapatinib and decreased with okadaic acid, combination treatment negated these changes and total Akt levels were significantly increased to similar levels to lapatinib alone treatment.

These changes indicate that the combination of PP2A inhibition and EGFR/HER2 inhibition have an enhanced effect at inhibiting survival signalling.

Table 3-2: Statistically significant alterations in HCC1954-L cells treated with lapatinib versus DMSO control by RPPA analysis.

DMSO v Lapatinib

Protein Phosphorylation site Fold change p value

EGFR - 0.37 0.009 GAB1 Y627 0.47 0.008 HER2 Y1248 0.74 0.016 HER3 Y1289 0.84 0.047 Akt2 - 1.40 0.034 Akt - 1.50 0.027 GSKβ - 1.64 0.029 MET - 2.13 0.040 PKCα - 2.43 0.016 NFκB p65 S536 2.48 0.007

Table 3-3: Statistically significant alterations in HCC1954-L cells treated with okadaic acid versus DMSO control by RPPA analysis.

DMSO v Okadaic acid

Protein Phosphorylation site Fold change p value

Akt2 - 0.11 0.0004

Src Y416 0.78 0.0350

Table 3-4: Statistically significant alterations in HCC1954-L cells treated with lapatinib plus okadaic acid versus DMSO control by RPPA analysis.

DMSO v Lapatinib + Okadaic acid

Protein Phosphorylation site Fold change p value

EGFR Y1068 0.41 0.003 MEK 1/2 S217 0.41 0.001 GAB1 Y627 0.49 0.017 EGFR Y1173 0.68 0.001 HER2 Y1248 0.73 0.003 mTOR - 0.74 0.042 HER3 Y1289 0.75 0.006 MAPK T202/Y204 0.76 0.027 Src Y416 0.76 0.016 Shc Y317 0.76 0.040 RAF - 0.78 0.003 mTOR S2448 0.79 0.019 VEGFR2 - 0.86 0.042 Akt - 1.39 0.048 NFκB p65 S536 2.99 0.001

Figure 3-14: Pathway analysis of HCC1954-L cells treated with lapatinib plus okadaic acid using PANTHER software.

3.10 Sensitivity of lapatinib resistant cell lines to LB-100

Although okadaic acid is a potent PP2A inhibitor, it is not safe as a therapeutic treatment [237, 282]. However, several PP2A inhibitors have been investigated in clinical trials. LB-100 (Lixte, Biotechnology), a therapeutic PP2A inhibitor, has recently been tested in a Phase I clinical trial in solid tumours and has been shown to enhance response to chemotherapy and radiotherapy in pre-clinial studies [196, 258, 259, 264]. Both lapatinib resistant cell lines and their parental cell lines were tested for sensitivity to LB-100 (Figure 3-15). Similar to PP2A inhibition with okadaic acid, SKBR3-L and HCC1954-L cell lines were significantly more sensitive to LB-100 compared to their respective parental cell lines. The LB-100 IC50 value for SKBR3-L

cells was 2.13 ± 0.20 µM compared to 5.38 ± 0.60 µM for SKBR3-Par cells. The LB- 100 IC50 values for HCC1954-L and HCC1954-Par cells were 2.31 ± 0.19 µM and

5.32 ± 0.82, respectively. Furthermore, lapatinib plus LB-100 was synergistic in the HCC1954-L and SKBR3-L cells (CI value at ED50 = 0.68 ± 0.26 and 0.56 ± 0.13,

Figure 3-15: Proliferation of (A) SKBR3-Par and SKBR3-L and (B) HCC1954- Par and HCC1954-L treated with LB-100 for 5 days was measured by acid phosphatase assay. Error bars represent standard deviation of biological triplicate experiments.

Figure 3-16: Proliferation of (A) SKBR3-L and (B) HCC1954-L treated with lapatinib, LB-100, or lapatinib plus LB-100 for 5 days was measured by acid phosphatase assay. Error bars represent standard deviation of biological triplicate experiments.