Identificación de biomarcadores de resistencia a taxanos en cáncer de cabeza y cuello. Estudio de alternativas terapéuticas

Abstract

Squamous cell carcinoma of the head and neck includes a heterogeneous group of neoplasms originated in the squamous epithelium of the upper aerodigestive tract. One of the therapeutic options for unresectable tumours diagnosed in locally advanced stages is induction chemotherapy with taxanes, platinums and 5-fluorouracil (TPF regimen) followed by radiotherapy. Despite the improvement in survival observed in recent years, there are subgroups of patients who present resistance to treatment and poor prognosis. For this reason, the main objective of this work was to characterize the molecular alterations associated with taxane-resistant head and neck squamous cell carcinoma. Paclitaxel resistance was induced in three cell lines derived from squamous carcinomas of the tongue (CAL33), larynx (32860) and oropharynx (32816) by step-wise dose increase, establishing two taxane-resistant cell lines (CAL33-R and 32816-R). However, the 32860 cell line did not acquire resistance to paclitaxel. This cell line presents a deletion in the terminal region of chromosome arm 3p that affects the ATG7 gene and causes an inhibition of autophagy. In order to determine whether this alteration was responsible for the lack of acquisition of paclitaxel resistance, the ATG7 gene was inactivated in the 32816 cell line using CRISPR-Cas9 technology, generating ATG7 knockout clones. In these clones, autophagy blockade, decreased sensitivity to paclitaxel and faster acquisition of paclitaxel resistance were observed. Thus, inactivation of ATG7 inhibits autophagy, but is not responsible for the differential behaviour observed in the 32860 cell line. The genomic profile of a series of 177 head and neck squamous cell carcinomas diagnosed at locally advanced stages was defined and the alterations found were associated with the response to the TPF regimen. Tumours from patients with toxicity to treatment presented a greater number of genomic changes. Losses in 4p were more frequent in this group, suggesting that this alteration could determine toxicity to TPF therapy. In addition, the region where ATG7 is located was analyzed and deletions were identified in 64.4% of the cases in the overall series, so this is a recurrent alteration in head and neck squamous cell carcinoma. Moreover, overexpression of ABCB1, encoding MDR1, a drug efflux pump, and underexpression of DNAJC15, encoding MCJ, a negative regulator of mitochondrial respiration, were identified in CAL33-R and 32816-R cell lines. These alterations were shown to be essential for taxane resistance in these cell lines and taken together could constitute a potential response biomarker. Phenotype characterization of CAL33-R and 32816-R cell lines revealed decreased proliferation and increased migratory capacity probably due to dysregulation of key signalling pathways in these processes. In addition, the role of extracellular vesicles in taxane resistance was analyzed. Differences in the protein content of vesicles released by the CAL33 and CAL33-R cell lines were identified, highlighting the possible transfer of resistance to other tumour cells using this mechanism. Finally, the response of taxane-resistant cell lines to other therapies was studied. These cell lines retained the response to 5-FU and showed increased sensitivity to DNA-damaging agents, including cisplatin and radiation. The reason for this cross-sensitivity is an increase in the levels of DNA damage together with alterations in DNA repair pathways. The response to olaparib, an inhibitor of DNA damage repair, was studied and it was observed that taxane-resistant cells also showed increased sensitivity to this drug, that should be considered as a therapeutic alternative. The results obtained suggest that ATG7 could condition taxane response in head and neck squamous cell carcinoma. Furthermore, ABCB1 overexpression together with DNAJC15 underexpression could be a biomarker of taxane response, determining the existence of resistance to treatment. The mechanism underlying this resistance could be based on an increase in mitochondrial ATP, production due to the loss of DNAJC15 expression, used by the efflux pump MDR1 to expel the drug, reducing its intracellular concentration and effect. The combination of DNA-damaging agents with DNA repair inhibidors could constitute an effective therapeutic alternative in taxane-resistant head and neck squamous cell carcinoma.