CYB5A and autophagy-mediated cell death in pancreatic cancer

Abstract

The highly invasive and chemoresistant phenotype of pancreatic cancer highlights the urgency to identify prognostic biomarkers and novel therapeutic targets. Recently, we observed a significant correlation between shorter survival and loss of the cytoband 18q22.3. Here we investigated genes encoded by this cytoband, and demonstrated the prognostic value of CYB5A in resected and metastatic patients. Furthermore, our in vitro and in vivo studies clarified CYB5A inhibitory activity of oncogenic phenotypes through autophagy induction. This raises the possibility that inhibition of CYB5A-deregulated downstream pathways, such as those involving TRAF6, may favor autophagy-mediated cancer cell death in selected subgroups of patients.

Pancreatic cancer is the fourth most common cause of cancer-related death. Prognosis is very poor with survival rates that have not improved over the past 40 years, and fewer than 5% patients remain alive 5 years after diagnosis.

More than 90% of exocrine pancreatic cancers are invasive pancreatic ductal adenocarcinomas (PDAC). The incidence of these tumors has increased steadily in the last decade and recent epidemiological studies predict rising mortality rates. The main reasons for the dismal prognosis include the lack of effective biomarkers for screening/diagnosis/prognosis, as well as the early metastatic spread and the intrinsic resistance to most currently available systemic treatments.

In recent years, detailed genetic analyses unraveled the pivotal mechanisms controlling pancreatic carcinogenesis, and further studies identified the substantial genomic heterogeneity of these tumors. In particular, cluster analysis of recurrently mutated genes defined 12 different core pathways, which lead to aberrant signaling in PDAC cells. Among these comprehensive genetic studies, we previously reported the association of genomic imbalances and clinical outcome using high-resolution array-comparative-genomic-hybridization in a cohort of 44 radically resected patients. In this series we observed a significant correlation between shorter survival and loss of the small cytoband 18q22.3, which contains 5 known genes (FBXO15, TIMM21/C18orf55, CYB5A, FAM69C/C18orf51, and CNDP2/CPGL).

In the present study we evaluated whether the mRNAs and/or proteins codified by the genes in the 18q22.3 cytoband were associated with the outcome in other independent cohorts of resected (N = 130) and metastatic (N = 50) patients. Low expression of CYB5A correlates with significantly shorter survival in all these cohorts, while the other 18q22.3-encoded genes exhibit no prognostic value.

Furthermore, we characterized and ascribed a novel function to CYB5A, autophagy induction. This was demonstrated by several in vitro and in vivo experiments, using gain- and loss-of-function models, through retrovirus-mediated upregulation and small interfering RNA, respectively. CYB5A is a membrane-bound cytochrome that reduces methemoglobin to ferrous hemoglobin, which is required for stearyl-CoA-desaturase activity. CYB5A functions also as an electron carrier for several membrane-bound oxygenases. This activity includes the reductive detoxification of carcinogen arylhydroxylamines. Therefore we hypothesized that CYB5A might affect PDAC aggressive behavior through modulation of DNA repair. However, models characterized by differential expression of CYB5A show a similar extent of double-strand break induction after exposure to 4-aminobiphenyl.

Conversely, the most striking effect of the CYB5A transduction is the markedly different morphological appearance of the cells, showing a round shape and a dramatic increase in cytoplasmic vacuolization. The cytoplasm and the nucleoplasm are slightly darker, although most nuclear structures do not carry signs of nuclear apoptosis, such as pyknosis or karyorhexis. All these features suggest an extensive induction of autophagy, and the ultrastructural analysis reveals both double-membrane vesicles containing different engulfed cytoplasmic contents, as well as autolysosomes. Quantitative analysis by morphometric methods confirms increased autophagic vacuole formation and accumulation of autolysosomes.

The ability of CYB5A to induce autophagy in PDAC cells is supported by the increase of acridine-orange-stained cells and by the double labeling of CYB5A and the autophagosome marker LC3-II, as well as by immunoblotting of the conversion of the 18-kDa precursor LC3-I to LC3-II. Furthermore, we observed a significant upregulation of several ATG genes, such as ATG5, ATG7, ATG9A, and ATG16L2. Based on these results, we performed a network analysis, revealing the potential physical interaction of CYB5A with TRAF6. This emerged as a molecular bridge for many diverse signals, both upstream and downstream, including AKT and MAP kinases, as well as with several genes involved in cell death, such as BCL2 and RPS6KB1. Remarkably, cells transduced with CYB5A show a significant reduction of TRAF6 expression, which is restored in rescue experiments by transfection with CYB5A siRNA.

In vivo, we demonstrated that the orthotopic models from the patient-derived CYB5A+ retrovirus-transduced cells have significantly reduced primary tumor dimension and metastatic spread, as monitored with bioluminescent-analysis of firefly- and Gaussia-luciferase signals, high-frequency ultrasound and magnetic resonance imaging. Moreover, CYB5A transduction significantly increases survival compared with control mice. These findings are associated with autophagy induction, as demonstrated by the significant increase in the expression of the autophagic markers LC3-II, ATG7, and ATG16L2. Additional immunohistochemical evaluation of the mouse tumors showed that the tumors in the CYB5A+ group had a weaker expression of TRAF6, associated with a significant increase of its turnover-regulator SQSTM1, further supporting the key role of this member of the TRAF family in the pro-autophagic signaling induced by CYB5A.

These findings add to the varied and often contradictory results regarding the activity of autophagy and its regulation in PDAC. Both decreased and increased autophagy are related to PDAC, and several lines of experimental evidence point at autophagy as a mechanism to protect PDAC cells under adverse environmental conditions, while other studies show that autophagy is detrimental to PDAC cells. From these conflicting data one could infer that the high inter- and intra-tumor heterogeneity in terms of complexity of genetic aberrations and resulting signaling pathway activations, which represents one of the main hallmarks of PDAC, might cause a range of different events involved in autophagy-mediated survival or death. While more work is needed to investigate how the different genetic/signaling features of PDAC regulate autophagic processes, the clear correlation of downregulation of CYB5A expression with shorter survival and the cross-talk between CYB5A and TRAF6 open new avenues for prognostic and therapeutic purposes. In particular, future studies aimed at inhibiting the CYB5A-deregulated downstream TRAF6 might provide a new tool favoring autophagy-mediated cancer cell death in subsets of PDAC patients stratified according to well-defined molecular markers.

Giovannetti E, Wang Q, Avan A, Funel N, Lagerweij T, Lee JH, Caretti V, van der Velde A, Boggi U, Wang Y, et al. Role of CYB5A in Pancreatic Cancer Prognosis and Autophagy Modulation. J Natl Cancer Inst. 2014;106:djt346. doi: 10.1093/jnci/djt346.