Targeting metabolism to improve immunotherapy in GSNOR-deficient colorectal cancer

Colorectal cancer (CRC) is the third most diagnosed type of cancer and the second leading cause of cancer death worldwide. Despite the increasing knowledge of CRC molecular biology and the development of new targeted therapies, its high heterogeneity hampers the efficacy of current treatments. Thus, there is a pressing need to identify new effective therapeutic targets and improve immune therapies for these patients. In this regard, S-nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumour suppressor role, although the mechanisms responsible are still largely unclear. Therefore, the main objective of this project was to understand the role of GSNOR in CRC tumorigenesis and its therapeutic implications. Firstly, we classified CRC tumours as GSNOR-high or low according to their GSNOR expression as assessed by immunohistochemistry (IHC). Accordingly, we found that GSNOR deficiency was associated with worse prognosis factors such as a larger tumour size at diagnosis, higher TNM stage, higher grade of tumour budding (TB), the CMS4 subtype, lower expression of the intestinal differentiation markers CDX2 and AE1/AE3 cytokeratin and a worse progression free survival (PFS) and overall survival (OS). We next investigated the differences in gene expression between CRC GSNOR-high and low tumours, uncovering significant alterations in metabolism and immune system pathways. Hence, GSNOR-deficient tumours were characterized by immune suppressive features and a dysregulation of their metabolism, favouring other metabolic pathways than OXPHOS. Total RNA from human CRC samples was extracted using the commercial RNeasy Mini Kit (Ref.: 74004, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Briefly, RNA from fresh‐frozen CRC tissues were obtained by mechanical disruption of 100 mg of tissue using gentleMACS™ M Tubes (Ref.: 130-096-335, Miltenyi Biotec, Bergisch Gladbach, Germany) and then lysed with 600 µL RLT buffer at 3,000 rpm for 10 min. at 4°C. Then, supernatants were centrifuged with 700µL of 70% ethanol using gDNA removal columns at 10,000 rpm for 1 minute at RT. Next, samples were washed with ethanol 70%. Samples were then washed with 700 µL RW buffer in RNA columns at 10,000 rpm for 1 minute at RT. Samples were washed twice with 500µL RPE buffer columns at 10,000 rpm for 1 minute at RT. Finally, RNA was collected using 30 µL RNase-free water at 10,000 rpm for 1 minute at RT. Finally, total RNA was quantified using the NanoDrop® 1000 spectrophotometer (NanoDrop® ND-1000 UV-Vis Spectrophotometer, NanoDrop Technology, Thermo Fisher Scientific, MA, USA), and RNA integrity Number (RIN) was measured using the Agilent 2200 TapeStation equipment (Agilent, CA, USA). After RNA was extracted as described above, a quality check was performed using TapeStation Analysis Software A.02.02 (SR1) (Agilent Technologies, CA, USA). A series of 34 CRC human samples with an RNA RIN ≥ 7 were selected and 100 ng were sent to Centre for Genomic Regulation (CRG, Barcelona, Spain) to obtain RNA libraries. The RNA libraries obtained were composed of short fragments of 50bp with paired ends and sequenced by using the NextSeq2000 sequencing system (100 cycles), performing 40 million reads/sample.