The Polo-like kinase 1 inhibitor onvansertib represents a relevant treatment for head and neck squamous cell carcinoma resistant to cisplatin and radiotherapy

Abstract

Rationale: Head and neck squamous cell carcinoma (HNSCC) represent the 4th most aggressive cancer. 50% of patients relapse to the current treatments combining surgery, radiotherapy and cisplatin and die two years after the diagnosis. Elevated expression of the polo-like kinase 1 (Plk1) correlated to a poor prognosis in epidermoid carcinomas. Methods: The molecular links between Plk1 and resistance to cisplatin/radiotherapy were investigated in patients and cell lines resistant to cisplatin and/or to radiotherapy. The therapeutic relevance of the Plk1 inhibitor onvansertib, alone or combined with cisplatin/radiotherapy, was evaluated on the proliferation/migration on HNSCC cell lines, in experimental HNSCC in mice, in a zebrafish metastasis model and on patient-derived 3D tumor sections. Results: Plk1 expression correlated to a bad prognosis in HNSCC and increased after relapse on cisplatin/radiotherapy. Onvansertib induced mitotic arrest, chromosomic abnormalities and polyploidy leading to apoptosis of sensitive and resistant HNSCC cells at nanomolar concentrations without any effects on normal cells. Onvansertib inhibited the growth of experimental HNSCC in mice and metastatic dissemination in zebrafishes. Moreover, onvansertib combined to cisplatin and/or radiotherapy resulted in a synergic induction of tumor cell death. The efficacy of onvansertib alone and in combination with reference treatments was confirmed on 3D viable sections of HNSCC surgical specimens. Conclusions: Targeting Plk1 by onvansertib represents a new strategy for HNSCC patients at the diagnosis in combination with reference treatments, or alone as a second line treatment for HNCSCC patients experiencing relapses.

Trial registration: ClinicalTrials.gov NCT01721876 NCT03303339 NCT03829410 NCT03414034.

Keywords: Head and neck squamous cell carcinoma (HNSCC); Plk1; cisplatin resistance; onvansertib; radiation resistance.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

© The author(s).

Figures

Figure 1

Plk1 is overexpressed in HNSCC patients and is associated with a poor prognosis. The tumors of HNSCC patients were analyzed for Plk1 mRNA levels (z-score). These results are in whole based upon data generated by the TCGA Research Network. (A) Comparison of Plk1 mRNA levels in several cancer. (B) Comparison of Plk1 mRNA levels between healthy (n = 44) and HNSCC tissues (n = 519). (C-D) The levels of Plk1 mRNA in tumors of HNSCC patients correlated with DFS (C) and OS (D). (E-F) The levels of Plk1 mRNA in patients' tumors from Centre Antoine Lacassagne correlated with DFS (E) and OS (F). DFS and OS were calculated from patient subgroups with mRNA levels that were less or greater than the third quartile value. Statistical significance (p value) is indicated. (G) Plk1 mRNA measured by qPCR in tumors from Centre Antoine Lacassagne's patients at diagnosis and after relapse to usual treatments (n = 20).

Figure 2

Characterization of CAL33 sensitive and resistant cell lines. (A) Plk1 immunoblotting on keratinocytes (kerat), human fibroblasts (FHN), CAL27 and CAL33 cells. GAPDH served as a loading control. (B) Clonogenicity assay of CAL33, CAL33 cis-R, CAL33 rad-R and CAL33 RR after 4 Gy of radiation (n = 2). (C) Cell viability of CAL33, CAL33 cis-R, CAL33 rad-R and CAL33 RR after 48 h of cisplatin treatment (3 and 10 µM) (n = 3). (D-E) Cell migration using Boyden's chamber assay on CAL27 and CAL33 sensitive and resistant cell lines. Results are expressed as the percentage of the control (n = 3). (F-G) Spheroids' invasion using 3D culture cell assays on sensitive and resistance CAL33 and CAL27 cells (n = 2). Results are expressed as arbitrary units (day 0 used as the control). Statistics were performed using ANOVA test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Onvansertib effects on sensitive and resistant CAL33 cell lines. (A) Clonogenicity assay of sensitive and resistant CAL33 cell lines after onvansertib (10 nM and 25 nM) or cisplatin (3 μM) treatment (n = 2). (B) Hematoxylin eosin saffron staining of sensitive and resistant CAL33 cell lines treated with onvansertib (25 nM) for 24 h. Abnormal mitosis was indicated with an arrow. (C-D) Cells were treated with onvansertib (25 nM, 50 nM or 100 nM) for 24 h. Cell cycle was measured by flow cytometry (n = 3) (C), G2/M arrest was evaluated by phospho-NPM and, phospho-H3 immunoblotting. HSP90 served as a loading control (n = 2) (D). (E-F) Polyploidy (number of nuclei 8N and 16N) was assessed by flow cytometry (n = 3) (E). Representation of polyploidy in CAL33 RR (F). (G) Cells were treated with onvansertib (25 nM and 50 nM) for 48 h. Cell death was evaluated by flow cytometry. Cells were stained with propidium iodure (PI) and Annexin V (AV). Histograms show AV+/PI- cells (early apoptosis) and AV+/PI+ cells (late-apoptosis or another cell death) (n = 3). (H) Immunostaining of PARP expression on CAL33 cell lines after exposure to onvansertib (100 nM) for 24 h. HSP60 served as loading control (n = 2). (I-J) Invasion of sensitive and resistant CAL33 cells using 3D cell culture assay (spheroids) after 6 days of onvansertib treatment (100 nM) grown in (I). Results are represented as arbitrary units (the no-treatment condition was used as a control) (n = 2) (J). Statistics were analyzed using ANOVA tests: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Onvansertib inhibits the growth of experimental tumors in mice. 106 CAL33 or CAL33 RR cells were subcutaneously injected in the flank of nude mice. When the tumors reached 100 mm3, mice were treated with onvansertib (60 mg/Kg) or with cisplatin (4 mg/Kg). (A) The tumor volume was measured twice a week. (B) At the end of the experiment, the weight of tumors has been evaluated and representative pictures of tumors treated or not with onvansertib are shown. (C) Necrosis was quantified in control and onvansertib-treated tumors (HES staining). (D) Pictures and quantification of proliferative cells measured by KI-67 staining. (E) Pictures of blood vessels and quantification evaluated by IHC of αSMA. (F-H) Murine VEGFA (mVEGFA, F), αSMA (mαSMA, G) and CD31 (mCD31, H) mRNA levels in tumors were determined by qPCR. Results are expressed as percent of control. Statistics were performed using an unpaired Student's t test: * p < 0.05, ** p < 0.001, **** p < 0.0001.

Figure 5

Onvansertib inhibits the formation and growth of CAL33 RR metastases in zebrafish. (A-D) Zebrafish embryos (n = 28) were injected with CAL33 RR (labelled in red) into the perivitelline space and immediately treated with onvansertib (50 nM) for 72 h. Representative images of zebrafishes are shown at 24 h, 48 h and 72 h (A). Quantification and representative images of no, few and high local metastases in the head after 72 h of treatment (B). Percentage of zebrafishes with tumor cells in the tail (distant metastasis) at 24 h were quantified (C). Area of distant metastases at 72 h were quantified (D). (E-F) Zebrafish embryos (n = 48) were injected with CAL33 RR (labelled in red) into the perivitelline space. 24 h later, zebrafish embryos with metastases were treated with onvansertib (50 nM) for 48 h. Representative image of zebrafish are shown (E) and variations of the metastases area before and after treatment were quantified (F). Statistics were performed using an unpaired Student's t test: ** p < 0.01, *** p < 0.001.

Figure 6

Onvansertib effects in combination with reference treatments (cisplatin and radiotherapy) on sensitive and resistant CAL33 cell lines. (A) Cells were treated 72 h with onvansertib (25 nM) in combination with cisplatin (3 µM) and radiotherapy (8 Gy). Cell death was evaluated by flow cytometry (n = 3). Cells were stained with Propidium Iodure (PI) and Annexin V (AV). Histograms show AV+/PI- cells (early-apoptosis) and AV+/PI+ cells (late-apoptosis or another cell death). (B-C) Cells were treated with onvansertib (2.5 nM), cisplatin (1 µM) and radiotherapy (2 Gy). Quantification (B) and representative image of clonogenicity assay (C) (n = 3). (D-E) 3D cell culture assay (spheroid) was treated with the combination of the three treatments: onvansertib (25 nM and 50 nM), cisplatin (3 µM) and radiation (2 Gy) and evaluated after 6 days. Quantification (D) and representative image of invasion assay (E) (n = 2). Statistics were analyzed using ANOVA test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and unpaired Student's t test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 7

Onvansertib on human tumor sections from HNSCC patients. (A) Tumors from HNSCC patients surrounded by agarose and tumor sections were done using a vibratome. These sections were cultured in a specific medium and treated for 4 days with onvansertib or/and cisplatin or/and radiotherapy. Analyses were then performed (n = 6). (B) Necrosis of biopsies' sections assessed using HES (Hematoxylin Eosin Saffran) staining. (C-I) Biopsies' sections treated with onvansertib (50 nM and 100 nM), cisplatin (3 μM and 10 μM) or radiotherapy (2 Gy) (C, E and H), or treated with onvansertib alone or in combination with cisplatin and radiotherapy (D, F and H). (C-D) ATP quantification of biopsies' sections. (E-G) Quantification of cell death by apoptosis by TUNEL assays on biopsies' sections. (H-I) Pictures and quantification of cell proliferation evaluated by KI-67 staining. Statistics were performed using ANOVA test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and unpaired Student's t test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.