Mutations of the LIM protein AJUBA mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell-cycle inhibitors

Abstract

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA, SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G₂/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 and Bora protein expression were decreased. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors.

Keywords: AJUBA; CHK1; Head neck squamous cell carcinoma; Polo-like kinase 1; WEE1.

Fig. 1

HNSCC cell lines have in vitro diverse sensitivities to drugs that affect mitotic progression. Fifty-nine HNSCC cell lines were treated with volasertib, AZD1775, or AZD7762 at seven concentrations ranging from 0.018 to 9.613 μM for 72 h, and their viability was estimated using a CellTiter-Glo assay. (A) Representative dose-response curves for cell lines sensitive and resistant to the drugs. (B) Distributions of the IC₈₀ values for the 59 cell lines. The vertical orange line is the C_max values for each drug.

Fig. 2

Inhibition or knockdown of PLK1 expression leads to cell-cycle arrest and apoptosis in HNSCC cell lines. HNSCC cells with different levels of sensitivity to treatment with the PLK1 inhibitor volasertib were treated with the drug at 50 nM or transfected with a PLK1 siRNA as indicated in the figures. (A and B) HNSCC cell-cycle stages determined according to 7-aminoactinomycin D and BrdU incorporation. (C) Western blots confirming the knockdown efficiency of PLK1 siRNA in the HNSCC cells. (D and E) HNSCC-cell apoptosis as measured using terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Western blotting for cleaved poly(ADP-ribose) polymerase (PARP). OSC19 cells incubated with 10 μM cisplatin for 48 hours were included as positive controls for apoptosis assessment.

Fig. 3

Mutations that predict sensitivity of HNSCC cells to treatment with PLK1, CHK1/2, and WEE1 inhibitors in vitro and in vivo. Drug sensitivity was compared with mutational status for all identified mutations in the 59 HNSCC cell lines (see Materials and Methods for details on mutation calling) for volasertib (A), AZD1775 (B), and AZD7762 (C). The box plots in A–C show the mutations that both correlated with drug sensitivity and were among the 50 top genes mutated in HNSCC cells. (D) IC₈₀ values for AZD1775, AZD7762, and volasertib with and without RAS (HRAS + KRAS) mutations in the HNSCC cell lines. (E–G) Mice bearing tongue tumors generated by injection of OSC19-luc cells were given 30 mg/kg volasertib weekly or a vehicle control. Their tumor sizes and bioluminescence were measured twice weekly (F), with representative photographs of the mice shown in E. Individual tumors are graphed as thin lines with markers. The mean tumor size is indicated by the thick solid line, and the standard deviation is indicated in dark gray. (G) Kaplan-Meier survival curves for the mice.

Fig. 4

AJUBA mutation and overexpression affect PLK1 and Bora protein expression and PLK1 inhibition-induced apoptosis in HNSCC cells. Western blots of AJUBA-mutant and AJUBA-wild-type (wt) HNSCC cell lines that were unsynchronized (A) or synchronized (E) with 100 ng/ml nocodazole for 16 h. The adjacent box plots show quantification of protein expression by the ImageJ software program and normalized to GAPDH. A pair of isogenic PCI15B cells transfected with a vector alone or a vector expressing full-length AJUBA (B) were incubated with volasertib at the indicated concentrations for 48 h, and apoptosis of the cells was measured using terminal deoxynucleotidyl transferase dUTP nick-end labeling alone (C) or for 30 min followed by Western blotting to measure PLK1 substrate inhibition (D). Phosphorylated MYT1 (pMYT1) expression was quantitated using the ImageJ software program and normalized according to their corresponding total levels of expression. OE, overexpression. *p < 0.05.

Fig. 5

AJUBA overexpression leads to cell-cycle progression in HNSCC cells. (A) A pair of isogenic PCI15B cell lines transfected with AJUBA or a vector alone was incubated with 100 ng/ml nocodazole for 16 h and then released into complete medium without any drugs. At the indicated time points, cells were incubated with BrdU and 7-aminoactinomycin D and subjected to fluorescence-activated cell sorter analysis to estimate the portion of cells in each phase of the cell cycle. OE, overexpression. (B) The two cell lines were plated sparsely and allowed to grow on plastic for 14 days before quantification. The assay was repeated and normalized according to the control (vector only); the mean ± standard deviation number of cell colonies is shown. *P < 0.05. (C) HNSCC cell-doubling times in AJUBA-wild-type (wt) and AJUBA-mutant (Mut) HNSCC cell lines.