EUKARYOTIC TRANSLATION INITIATION ACTORS (EIFS) AS NOVEL BIOMARKERS IN HEAD AND NECK SQUAMOUS CELL CARCINOMA (HNSCC)

Abstract

The present invention relates to a method of diagnosing Head and Neck Squamous Cell Carcinoma (HNSCC) in an individual. In addition, the present invention relates to a method of providing a survival prognosis to an individual suffering from Head and Neck Squamous Cell Carcinoma (HNSCC). Moreover, the present invention relates to a kit for performing the above-mentioned methods.

Claims

1. A method of diagnosing Head and Neck Squamous Cell Carcinoma (HNSCC) in an individual (suspected of having HNSCC) comprising the step of: determining the level of at least one eukaryotic Initiation Factor (eIF), wherein the at least one eIF is selected from the group consisting of eIF2S1, eIF2S2, eIF2S3, eIF2B1, eIF2B2, eIF2B3, eIF2B4, and eIF2B5, and/or the level of at least one eIF kinase, wherein the at least one eIF kinase is selected from the group consisting of eIF2AK1, eIF2AK2, eIF2AK3, and eIF2AK4, in a biological sample from an individual (suspected of having HNSCC).

2. The method of claim 1, wherein the level of the at least one eIF is compared to a reference level of said at least one eIF.

3. The method of claim 2, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one healthy subject, and wherein the level of the at least one eIF above the reference level indicates that the individual suffers from HNSCC.

4. (canceled)

5. The method of claim 1, wherein the level of the at least one eIF kinase is compared to a reference level of said at least one eIF kinase.

6. The method of claim 5, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one healthy subject, and wherein the level of the at least one eIF kinase below the reference level indicates that the individual suffers from HNSCC.

7. (canceled)

8. A method of providing a survival prognosis to an individual suffering from Head and Neck Squamous Cell Carcinoma (HNSCC) comprising the step of: determining the level of at least one eukaryotic Initiation Factor (eIF), wherein the at least one eIF is selected from the group consisting of eIF2S1, eIF2S2, eIF2S3, eIF2B1, eIF2B2, eIF2B3, eIF2B4, and eIF2B5, and/or the level of at least one eIF kinase, wherein the at least one eIF kinase is selected from the group consisting of eIF2AK1, eIF2AK2, eIF2AK3, and eIF2AK4, in a biological sample from an individual (suspected of having HNSCC).

9. The method of claim 8, wherein the level of the at least one eIF is compared to a reference level of said at least one eF.

10. The method of claim 9, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one subject suffering from HNSCC, and wherein the level of the at least one eIF comparable with or above the reference level indicates that the individual has a poor survival prognosis, or the level of the at least one eIF below the reference level indicates that the individual has a good survival prognosis.

11. (canceled)

12. The method of claim 8, wherein the level of the at least one eIF kinase is compared to a reference level of said at least one eIF kinase.

13. The method of claim 12, wherein the reference level is the level determined by measuring at least one reference biological sample from at least one subject suffering from HNSCC, and wherein the level of the at least one eIF kinase comparable with or below the reference level indicates that the individual has a poor survival prognosis, or the level of the at least one eIF kinase above the reference level indicates that the individual has a good survival prognosis.

14-17. (canceled)

18. The method of claim 1, wherein the level of the at least one eIF is determined by measuring mRNA or protein levels.

19. The method of claim 1, wherein HNSCC is selected from the group consisting of Neoplasm Clinical Primary Tumor Stage (T), Neoplasm Clinical Regional Lymph Node Stage (N), and Neoplasm Clinical Distant Metastasis Stage (M).

20.-21. (canceled)

22. A kit for diagnosing Head and Neck Squamous Cell Carcinoma (HNSCC) in an individual (suspected of having HNSCC) or for providing a survival prognosis to an individual suffering from HNSCC, wherein said kit comprises means for determining the level of at least one eukaryotic Initiation Factor (eIF), wherein the at least one eIF is selected from the group consisting of eIF2S1, eIF2S2, eIF2S3, eIF2B1, eIF2B2, eIF2B3, eIF2B4, and eIF2B5, and/or the level of at least one eIF kinase, wherein the at least one eIF kinase is selected from the group consisting of eIF2AK1, eIF2AK2, eIF2AK3, and eIF2AK4, in a biological sample from an individual.

23. The kit of item 22, wherein HNSCC is selected from the group consisting of Neoplasm Clinical Primary Tumor Stage (T), Neoplasm Clinical Regional Lymph Node Stage (N), and Neoplasm Clinical Distant Metastasis Stage (M).

24.-33. (canceled)

34. A method for treating HNSCC in an individual comprising the step of: administering (an effective amount of) an eIF modulating compound to an individual in need thereof, wherein the at least one eIF is selected from the group consisting of eIF2S1, eIF2S2, eIF2S3, eIF2B1, eIF2B2, eIF2B3, eIF2B4, and eIF2B5.

35. The method of claim 34, wherein the eIF modulating compound is an eIF binding/inhibiting molecule or an eIF molecule.

36. The method of claim 35, wherein (i) the eIF binding/inhibiting molecule is selected from the group consisting of an anti-RNA, a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a polypeptide or peptide, and an antibody or an antibody fragment thereof, or (ii) the eIF molecule is a nucleic acid molecule encoding the eIF.

37. The method of claim 34, wherein in addition to an eIF modulating compound, a drug different from the eIF modulating compound is administered.

38. The method of claim 37, wherein the drug different from a eIF modulating compound is selected from the group consisting of a drug used in cancer therapy, immunotherapy, chemotherapy, hormone therapy, gene therapy, and infectious therapy.

39. The method of claim 37, wherein HNSCC is selected from the group consisting of Neoplasm Clinical Primary Tumor Stage (T), Neoplasm Clinical Regional Lymph Node Stage (N), and Neoplasm Clinical Distant Metastasis Stage (M).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0208] The following Figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

[0209] FIG. 1: Shows a Kaplan-Meier analysis of survival functions. High mRNA Expression of EIF2S1/eIF2a correlated with low survival prognosis. Light grey=low mRNA expression of EIF2S1/eIF2, dark grey=high mRNA expression of EIF2S1/eIF2.

[0210] FIG. 2: Shows that mRNA expression of EIF2S1/eIF2a is prognostic relevant (Spearman correlation rank)

[0211] FIG. 3: Shows that EIF2S1/eIF2a is upregulated in HNSCC (3A immunostaining, 3B diagram with results of immunostaining).

[0212] FIG. 4: Shows that treatment with EIF2S1/eIF2a inhibitor Salubrinal leads to a reduction in cell viability.

[0213] FIG. 5: Shows that EIF2S1/eIF2a inhibitor Salubrinal leads to reduction of cell viability in 3D-cell culture.

EXAMPLES

[0214] The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Methods

Bioinformatics

[0215] A multivariate analysis of the expression data of 279 HNSCC casesdata were available via cBioPortal.comwas performed. Patients were divided into two groups with high and low mRNA expression of the following genes: EIF2A, EIF2S1, EIF2S2, EIF2S3, EIF2AK1, EIF2AK2, EIF2AK3, EIF2AK4, EIF2B1, EIF2B2, EIF2B3, EIF2B4, EIF2B5. The overall survival of both groups was compared with the log-rank test. p<0.05 was considered to be significant.

Immunohistochemistry

[0216] Immunohistochemical tests were performed using an ultraView Universal DAB detection kit (Ventana Roche) on an automated immunostaining device (BenchMark Ultra, Roche}. From tissue fixed in formalin and embedded in paraffin thin sections of 4 m were prepared, deparaffinated, rehydrated and subjected to a heat-induced antigen demasking. The cuts were then pre-conditioned according to the Manufacturer's instructions and wrapped with Canada Balsam.

[0217] The sections were then evaluated by two independently acting pathologists (S. S. and R. M.} by light microscopy. The intensity score was determined by comparison with the staining of a control: 0=no staining; 1=weak staining; 2=moderate staining; 3=strong staining. The proportional score corresponded to the percentage of stained tumor cells: 0% O; <20% 1; 21-50% 2; 51-80%3; >80%4. The total immune-staining score was calculated by multiplying the two values. In total, 74 HNSCC samples were examined. The tumors were taken from the following sites: Larynx, hypopharynx, oropharynx, epipharynx, oral floor, palate, tonsilla, tongue and base of tongue. 24 samples of appropriate non-tumorous tissue were tested for comparison. The median values of IS, PS and TIS for normal tissue and the HNSCC samples were compared using the Kruskal-Willis test. The significance level was set at p<0.05.

MTT Assay in Cell Culture

[0218] Cell viability assays were carried out with 3 moderately differentiated laryngeal cancer cell lines (SNU-899, SNU-1066, SNU-1214}. MTT reagent stock solution was prepared by dissolving 5 mg crystal in lml PBS and filter sterilization. The cell culture medium was aspirated and replaced by 100 l fresh medium and 10 l MTT stock solution per well. The plates were incubated at 37 C. for two hours. The supernatant was removed and 100 l DMSO was added to each well. After 15-minute incubation at room temperature in a orbital shaker the absorption at 560 m was detected. All measurements were performed three times using GloMax@-Multi+Microplate Multimode Reader (Promega, Germany}. All experiments were performed three times with three repetitions.

Patient Derived 3D-Organoids (PD30} and Chemosensitivity Testing

[0219] Tumor samples from patients, who have undergone resection in the ENT clinic of Otto-von-Guericke University in Magdeburg, were incubated overnight in the RPM medium containing penicillin, streptomycin and amphotericin. On the following day, the tissue samples were crushed, passed through a sieve set to produce a size fraction of between 30 and 100 m, which were then incubated on 24-well plates at 37 C. The PD30s were incubated with Salubrinal in a 384-well culture plate for 72 h (i.e. the double population doubling time}. Thereafter, the CellTiter-Glo luminescence cell viability assay was performed. The reagent mixture was assembled according to the manufacturer's instructions. All experiments were performed three times with three repetitions

Results

[0220] mRNA expression data of 279 HNSCC patients were analysed and for patients with strong/high expression of all subunits of eIF2 (i.e. EIF2S1, EIF2S2, EIF2S3, EIF2B1, EIF2B2, EIF2B3, EIF2B4, and/or EIF2B5) and low expression of the eIF2 kinases (i.e. EIF2AK1, EIF2AK2, EIF2AK3, and/or EIF2AK4) a lower overall survival was found. It was also found that all subunits of eIF2 (i.e. EIF2S1, EIF2S2, EIF2S3, EIF2B1, EIF2B2, EIF2B3, EIF2B4, and/or EIF2B5) were upregulated in HNSCC patients compared to healthy controls and that eIF2 kinases (i.e. EIF2AK1, EIF2AK2, EIF2AK3, and/or EIF2AK4) were downregulated in HNSCC patients compared to healthy controls. In particular, applying immunohistochemistry, an overexpression of EIF2S1 compared to non-tumorous tissue was shown. (p=0.039). Then, the effect of Salubrinal on the cell viability of cancer cells of the moderately differentiated SCC of the larynx was investigated. Incubating the cell culture with the active ingredient a 20-40% reduction of the viability of the cells was found, whereby the effect was dependent on the cell line. This effect was dependent on the dosage and the highest efficacy was observed at a dosage of 50 M after 72 hours. Similarly, treating 3D-organoids of HNSCC derived from patient populations with the active substance a dose-dependent reduction of cell viability (average IC50 of 56 M (in the range of between 16-158 M) was found in almost all samples.

[0221] Thus, all subunits of eIF2 (i.e. EIF2S, EIF2S2, EIF2S3, EIF2B1, EIF2B2, EIF2B3, EIF2B4, and/or EIF2B5) and eIF2 kinases (i.e. EIF2AK1, EIF2AK2, EIF2AK3, and/or EIF2AK4) are useful diagnostic markers as well as a therapeutic targets for HNSCC.