GNA13 expression promotes drug resistance and tumor-initiating phenotypes in squamous cell cancers

Abstract

Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess tumor-initiating (TIC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of TIC phenotypes. Many of the implicated GPCRs signal through the G protein GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in patients. GNA13 levels modulate drug resistance and TIC-like phenotypes in patient-derived head and neck squamous cell carcinoma (HNSCC) cells in vitro and in vivo. Blockade of GNA13 expression, or of select downstream pathways, using small-molecule inhibitors abrogates GNA13-induced TIC phenotypes, rendering cells vulnerable to standard-of-care cytotoxic therapies. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker for tumor progression, and that interfering with GNA13-induced signaling provides a novel strategy to block TICs and drug resistance in HNSCCs.

Fig. 1. GNA13 is a prognostic marker of survival in solid tumors

: Kaplan–Meier survival plot of a Head and neck cancer (HNSCC) patients comparing those expressing higher vs. lower than median GNA13 mRNA levels. The breakdown of number patients for High and Low GNA13 groups is indicated in the table below the graphs. b GNA13 staining of HNSCC tissue microarrays by immunohistochemistry (IHC) and a representative staining of sections negative (upper panel) and positive for GNA13 (lower panel). c Kaplan-Meier analysis showing distant metastases-free survival in patients that are positive vs. negative for GNA13 IHC staining. d Immunoblot for GNA12 and GNA13 in patient-derived HNSCC tumor cells. Tubulin is shown as loading control. e Table showing the names of the primary cell lines and clinical characteristics of the patient from which these cell lines were derived from

Fig. 2. GNA13 induces resistance to treatment with cytotoxic drugs and γ-irradiation in HNSCC.

a Basal GNA13 protein expression correlates to resistance to cytotoxic treatment-induced apoptosis in a panel of patient derived HNSCC cell lines. The cytotoxic treatment (x-axis)—induced apoptosis is shown as % Annexin V staining (y-axis) compared to untreated control (NC). See Fig.1E for the Immunoblot in the inset showings basal GNA13 expression.; Tubulin is used as control. b Knockdown of GNA13 in GNA13-high cells induces sensitivity while overexpression in GNA13-low cells induces resistance to cytotoxic treatment-induced apoptosis. Graphs show percentage of cells that underwent apoptosis (based on Annexin V staining (y-axis)) in response to cytotoxic treatment (x-axis) compared to negative controls (NC) after GNA13-knockdown in NCC-HN43 and NCC-HN19 and over-expression in NCC-HN26 and NCC-HN23 respectively. Immunoblot of GNA13 protein expression for each line is shown in the inset with tubulin as loading control. c Blocking GNA13 expression reduces the IC₅₀ values to cisplatin, while overexpression does the opposite. Assessment of IC₅₀ values from cell viability assays after cisplatin treatment in GNA13-knockdown (ShRNA-1 and ShRNA-2) NCC-HN43 and NCC-HN19 (compared to ShRNA-control), and GNA13-overexpression in NCC-HN26 and NCC-HN73 (compared to vector control) is shown. d GNA13 overexpression induces resistance to cisplatin-induced tumor regression in vivo. The volume of tumor xenografts in NOD/SCID mice using GNA13 overexpressing NCC-HN26 cells with/without cisplatin treatment, compared to vector control is shown (Top panel). Mice were treated with cisplatin (5 mg/Kg body weight) or vehicle control. Images of the harvested tumors after six doses of cisplatin or vehicle for NCC-HN26-vector and NCC-HN26-GNA13 expressing cells are shown (Lower panel). e GNA13 protein expression is higher in cisplatin resistant patient derived HNSCC cells. Assessment of IC₅₀ values from cell viability assays after cisplatin treatment in cisplatin resistant (CR) NCC-HN120 cells compared to parental (WT) primary or metastatic lines is shown. Immunoblot for GNA13 is shown in the inset with tubulin as loading control. For all graphs, p-values denoted as: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 and ****, p < 0.00005; error bars indicate standard deviation

Fig. 3. GNA13 promotes tumor initiating cell-like properties in HNSCC cells.

a Cells grown in 3D spheroid cultures show increased TIC markers and GNA13 protein expression compared to cells grown in monolayers. Graphs showing TIC sub-population using ALDH1 activity and CD44⁺/CD24⁻-cell fractions in NCC-HN43 and NCC-HN26 grown as monolayers (M) and spheroids (S) (with Western blots showing GNA13 levels) b Blocking GNA13 in GNA13-high cells abrogates TIC markers while overexpression in GNA13-low cells induces them. Relative ALDH1 activity and relative CD44⁺/CD24^- is shown (y-axis) for GNA13-knockdown cells NCC-HN43 (ShRNA-1 and ShRNA-2, compared to ShRNA-control) (Top panel), and GNA13-overexpressing NCC-HN26 cells compared to vector control (Lower panel). c Knockdown of GNA13 suppresses the number of sphere forming units (SFU) in NCC-HN43 cells while overexpression in NCC-HN26 cells induces the SFUs. Number of sphere forming units is plotted in y-axis and the number of cells seeded in each well is shown in x-axis. d, e GNA13 is essential for long-term self-renewal of HNSCC cells. Cell culture images showing sphere formation of primary, secondary and tertiary spheroids in 3D culture, with subsequent colony formation in monolayer culture (Crystal violet stain) of (d) GNA13-overexpressing NCC-HN26 cells (compared to vector control) and (e) GNA13-knockdown in NCC-HN43 cells (ShRNA-1 and ShRNA-2, compared to ShRNA-control). f GNA13 is essential for early tumor initiation and induces tumor size. Graph showing tumor growth curves for limiting dilution experiments, where GNA13-overexpressing NCC-HN26 and vector control cells were injected into flanks of NOD/SCID mice (n = 5 each); Number of cells injected were: 5 × 10⁵, 5 × 10⁴ and 5 × 10³ and tumor volumes were measured for 61 days. Xenograft tumors harvested after 61 days of injecting 5 × 10⁵ cells in NOD/SCID mice is shown for (g) GNA13 overexpressing NCC-HN26 compared to vector control cells and (h) GNA13-knockdown NCC-HN43 cells (compared to ShRNA-controls). All experiments are performed at least three times with three replicates (n = 3) and a representative figure is shown. For all graphs, p-values denoted as: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 and ****, p < 0.00005; error bars indicate standard deviation

Fig. 4. GNA13 does not induce Epithelial to Mesenchymal Transition (EMT) in HNSCC

A real-time PCR screen for EMT marker expression in 4 different HNSCC cells either with enforced expression (left panel) or knockdown (right panel) of GNA13 showed no correlation of GNA13 expression to EMT induction. A real-time PCR analysis for (a) Epithelial markers—E-cadherin (Ecad) and Claudin-1 (CLDN1), (b) Mesenchymal markers—Fibronectin-1 (FN1), N-Cadherin (Ncad) and Vimentin (VIM) and (c) Mesenchymal Transcription Factors (TFs)—Snail-1 (SNAI1), Snail-2 (SNAI2), Twist-1 (TWIST) and Zinc Finger E-Box Binding Homeobox 1 (ZEB1) was performed in HN26 and HN73 (low-GNA13) cells stably expressing either vector control or GNA13, and in HN43 and HN19 (high-GNA13) cells stably expressing ShRNA-control or two different ShRNAs targeting GNA13. The mRNA expression of each gene is shown in y-axis as relative expression to either vector controls or ShRNA-controls respectively. d A hierarchical clustering analysis of GNA13 mRNA expression to established EMT markers or Stemness markers showed that GNA13 expression correlates to stemness rather than EMT inducing genes. The blue bar represents low GNA13 and the red bar represents high GNA13 expressing patients and the heat map shows EMT (left panel) or stemness (right panel) related genes’ expression as a heat map generated using raw z-scores from the same patients. For all graphs, p-values denoted as: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 and ****, p < 0.00005; error bars indicate standard deviation

Fig. 5. GNA13 promotes TIC-like phenotype and drug resistance via NFκB and MAPK signaling pathways.

a GNA13 induces NFκB and AP-1 transcription factors’ activity in HNSCC cells. Graphs showing luciferase reporter activity driven by the following promoters: NFκB, AP-1, YAP/TAZ (TEAD), SRE and WNT-βCatenin (Top-Flash), in GNA13-knockdown NCC-HN43 (ShRNA-1 and ShRNA-2, compared to ShRNA-control), and GNA13 overexpressing NCC-HN26 compared to vector control cells. The ratio of relative light (RLU) units of the reporters to RLU of Renilla-Luciferase control is plotted as fold increase to pGL3-basic vector in y-axis. b GNA13 induces NFκB and JNK/MAPK/AP-1 signaling pathways. Immunoblots showing the effect of GNA13-knockdown or over-expression on phosphorylation of c-JUN, IκB and ERK1/2 in NCC-HN43 and NCC-HN26 cells (compared to ShRNA-Control or vector controls), respectively. Tubulin is shown as a loading control. c Blocking NFκB and/or JNK/MAPK/AP-1 signaling pathways abrogates GNA13-induced TIC markers. Graph showing effect of increasing amounts of JNK inhibitor SP6000125 (JNKi), NFκB inhibitor BAY-11-7082 (BAYi) and MEK1/2 inhibitor MEK162 (MEKi) on CD44⁺/CD24⁻—cell fractions in GNA13-knockdown NCC-HN43 (ShRNA-1 and ShRNA-2, compared to ShRNA-control) (top panel), and GNA13-overexpressing NCC-HN26 (compared to vector control) (lower panel). d Blocking NFκB and/or JNK/MAPK/AP-1 signaling pathways abrogates GNA13-induced Cisplatin resistance. Graph showing IC₅₀ for cisplatin after blocking JNK (JNKi), NFκB (BAYi) or MEK1/2 (MEKi) pathways on GNA13 overexpressing NCC-HN26 cells compared to vector control. For all graphs, p-values denoted as: *, p < 0.05, **, p < 0.005, ***, p < 0.0005 and ****, p < 0.00005; error bars indicate standard deviation