Fig 2: MAPK activation increases ERK negative feedback and activates STAT3(A) A simplified schematic showing the MAPK pathway and its negative feedback regulators DUSP6, SPRY2, and ETV5. Created with BioRender.com.(B) Western blot analysis on SCLC cell lines as indicated upon MEKDDS217D/S221D expression (0, 1, 2, 3 days). MEKDD, total and phosphorylated ERK1/2, DUSP6, SPRY2, ETV5 are shown with β-actin used as loading control. MEKDD, total and phosphorylated ERK1/2 were taken from Figure 1D for presentation purposes.(C) Heatmap of SCLC cell lines showing log2 fold change for relative change in gene expression after MEKDDS217D/S221D expression (24 h) for the indicated genes. Unpaired t test was performed between ASCL1- and non-ASCL1-SCLC cell lines with p value as indicated.(D) Western blot analysis on ASCL1-driven SCLC cell line H69 cultured as adherent, suspension, and mixed upon MEKDDS217D/S221D expression. Total and phosphorylated ERK1/2, DUSP6, and SPRY2 are shown with β-actin used as loading control. Total and phosphorylated ERK1/2 were taken from Figure 2C for presentation purposes.(E) Phosphokinase array in H720 and H82 upon MEKDDS217D/S221D expression after 24 h. Phosphorylated ERK1/2 (T202/Y204) and STAT3 (S727) are indicated.(F) Mean pixel density derived from (D) for phosphorylated ERK1/2 (T202/Y204) and STAT3 (S727) after 24 h of MEKDDS217D/S221D expression in the indicated cell lines (±SEM). Unpaired t test was performed so that ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.(G) Western blot analysis on SCLC cell lines as indicated upon MEKDDS217D/S221D expression (0, 1, 2, 3 days). Total and phosphorylated ERK1/2, total and phosphorylated STAT3, and total and phosphorylated AKT are shown with β-actin used as loading control. Total and phosphorylated ERK1/2 were taken from Figure 2B for presentation purposes.

Fig 3: SCLC-A is selectively sensitive to MAPK activation in vitro and in vivo(A) Bar graph showing absolute counts for Mutation, Fusion, Deep Deletion, Amplification, and Multiple Alterations found in MAPK pathway genes (KRAS, HRAS, BRAF, RAF1, MAP3K1, MAP2K1, MAP2K2, MAP2K4, MAPK1, DUSP4, and CCND1) comparing SCLC (n = 469) and NSCLC (n = 8,948) clinical samples. The dataset was derived from the MSK-IMPACT clinical sequencing cohort (Cerami et al., 2012; Gao et al., 2013) as of 24 March, 2021, with variants of unknown significance and germline mutation excluded. p Value derived from Pearson's chi-squared test considering all alterations.(B) Graph showing MAPK Pathway Activity Score (SPRY2, SPRY4, ETV4, ETV5, DUSP4, DUSP6, CCND1, EPHA2, and EPHA4) comparing SCLC (n = 53) and NSCLC (n = 133) cell lines (±SD). MPAS was calculated as in Wagle et al. (2018) using Z scores for each gene/cell line from CCLE. Unpaired t test was performed between SCLC and NSCLC so that ∗∗∗∗p < 0.0001.(C) Overview of SCLC cell lines and their subtype used in this study. ASCL1-driven (SCLC-A, red), NEUROD1-driven (SCLC-N, blue), and POU2F3-driven (SCLC-P, green).(D) Western blot analysis on ASCL1-driven (H510, H69, DMS79, H720, red), NEUROD1-driven (H82, blue), POU2F3-driven (H526, green) SCLC cell lines and NSCLC cell line A549 upon MEKDDS217D/S221D expression (0, 1, 2, 3 days). MEKDDS217D/S221D (HA-tag) and total and phosphorylated ERK1/2 are shown, and β-actin was used as loading control.(E) Cell viability assay with SCLC cell lines following MEKDDS217D/S221D expression for 9 days. Illustrated are the average relative changes (±SEM) of cell viability. Unpaired t test was performed between ASCL1-driven (DMS79, H69, H720, H510) and non-ASCL1-driven (H82, H526, A549) SCLC cells so that ∗∗∗∗p < 0.0001.(F) ASCL1-driven SCLC cell line H510-MEKDD was injected subcutaneously into athymic mice (n = 6/cohort) and checked biweekly for palpable tumors. Mice in the “MEKDD ON” cohort were kept on a doxycycline chow diet throughout the experiment. Graph illustrates average tumor volume (mm3, ±SEM) over time. Unpaired t test was performed between the two cohorts at the indicated time point so that ∗∗p < 0.01, ∗∗∗p < 0.001.(G) Overall survival of the recipient mice from F is plotted as a Kaplan-Meier curve and denoted as the time from injection of cells to tumor volume of 1,500 mm3. p Value was calculated by log rank test.(H) NEUROD1-driven SCLC cell line H82-MEKDD was injected subcutaneously into athymic mice (n = 6/cohort) and checked biweekly for palpable tumors. Mice in the “MEKDD ON” cohort were kept on a doxycycline chow diet throughout the experiment. Graph illustrates tumor volume (mm3, ±SEM) over time. Unpaired t test was performed between the two cohorts at the indicated time point so that ∗p < 0.05, ns > 0.05. (I) Overall survival of the recipient mice from H is plotted as a Kaplan-Meier curve and denoted as the time from injection of cells to tumor volume of 1,500 mm3. p Value was calculated by log rank test.

Fig 4: miR-22-3p indirectly upregulates SPRY2 by inhibiting the expression of CBL.A. qPCR of SPRY2 in HepG2 mock cells and HepG2 overexpressing miR-22-3p cells. B. Immunoblotting of SPRY2 in the four cell lines: HepG2 mock cells, HepG2 overexpressing miR-22-3p cells, HepG2 control cells, and miR-22-3p knockdown HepG2 cells. C. Ubiquitination level of SPRY2 in the above four cell lines.

Fig 5: miR-22-3p suppresses ERK signaling by upregulating the expression of SPRY2.A and B. Immunoblotting of ERK and p-ERK in HepG2 mock cells, HepG2 overexpressing miR-22-3p cells, and HepG2 knockdown miR-22-3p cells. C. Immunoblotting of ERK and p-ERK in HepG2 mock cells, HepG2 overexpressing miR-22-3p cells, and HepG2 overexpressing miR-22-3p/ SPRY2 knockdown cells. D. miR-22-3p/CBL/SPRY2/ERK axis regulates EMT, migration and invasion of liver cancer cells.