although the sh - sy5y cell line is a subclone of the sk - n - sh cell line, the global gene expression microarray showed a profound differential expression of genes in these two cell lines [ 60 ]. however, the mechanism involved is unclear. in the present study, we noted that myt1l was up - regulated in the sh - sy5y cell line at both mrna and protein levels ( figure 3a, b ), while in its parental line, sk - n - sh, only myt1l mrna was up - regulated. the myt1l protein was undetectable, which may implicate the involvement of post - transcriptional regulation, such as mirna ( s ), in myt1l expression. some of the molecules analyzed in this manuscript can be molecular targets in cancer therapy. the il - 8 - cxcr1 / 2 axis has been proposed as a potential therapeutic target for numerous cancers due to its contributing roles in proliferation, migration / invasion, angiogenesis, and tumor immunosupp ression [ 61, 62 ]. the tumor - produced il - 8 is a potent chemoat tractant that recruits cxcr1 / 2 - expressing human myeloid - derived suppressor cells to tumor foci, playing a crucial role in immune resistance [ 63 ]. il - 8 receptor cxcr1 may be a biomarker for cancer stem cells [ 64 ], including glioblastoma stem cells [ 58 ]. targeting myeloid cell cxcr1 / 2 enhances antitumor immunity in pancreatic cancer [ 65 ]. neutralizing il - 8 or inhibiting its receptor cxcr1 / 2 potentiates anti - pd - 1 - mediated antitumor immunotherap y for glioma [ 66 ]. these findings highlight il - 8 and cxcr1 / 2 as potential therapeutic targets for cancer. mcclelland and colleagues have summarized numerous inhibitors developed to target il - 8 or its receptor cxcr1 / 2 for the treatment of chronic obstructive pulmonary disease ( copd ), asthma, diabetes, pneumonia, and solid tumors, including prostate cancer [ 62 ]. phase 1 and 2 clinical trials in prostate cancer with navarixin, a small - molecule inhibitor of cxcr1 / 2, have been completed. phase 1 and 2 clinical trials in prostate cancer with bms - 986253, a monoclonal antibod y against il - 8, are still active. however, none of these inhibitors have yet been approved to treat cancer. the erk1 / 2 signaling pathway contributes to numerous biological and pathological processes. excessive activation of the erk1 / 2 pathway is a hallmark of human malignancies [ 67, 68, 69, 70, 71, 72, 73 ] due to its crucial role in maintaining sustained cellular proliferation, resistance to cell death, angiogenesis, invasion, and metastasis. many fda - approved drugs target upstream regulators of the erk1 / 2 pathway, resulting in an indirect inhibition of erk1 / 2 kinase [ 74 ]. erk1 / 2 signaling may be reactivated upon the development of drug resistance. liu and colleagues have described 10 small - molecule inhibitors of erk1 / 2 which were developed recently and have undergone clinical trials in cancer patients [ 75 ]. some of them have shown promising results in the treatment of cancers. surprisingly, a large body of evidence also indicates a contributing role of erk1 / 2 activation in cancer cell apoptosis primarily induced by chemical compounds, including glioblastoma, bladder, breast, colon, endometrial, head and neck, lung, renal cell, and testicular germ cell carcinomas [ 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 ], and mechanically, through activation of caspase 3 and induction of reactive oxygen species. both oncogene and non - oncogene addiction have been demonstrated as fantastic targets for cancer therapy due to their crucial role in supporting cancer progression [ 86 ]. using a unique glioblastoma cell model system, m059j with deficient dna - pk, while m059k with normal dna - pk, we demonstrate in vitro that myt1l - overexpressing cells show non - oncogene addiction to dna - pk. genomic instability due to defects in dna repair genes is a key hallmark of cancer that drives the development of human malignancies [ 87 ]. however, these defects may also offer cancer cells opportunities to evolve a dependency on a non - oncogene addiction gene. for instance, brca2 - deficient cancer cells are more dependent on parp1 to repair dna for survival, resulting in an increased sensitivity to parp1 inhibitors [ 88 ], which kill the cancer cells through the genetic concept of synthetic lethality [ 89 ]. targeting mediators of dna repair has become a state - of - the - art strategy for cancer [ 90 ], leading to the development of inhibitors of key mediators of dna repair, including dna - pk. interestingly, genetic and pharmacological targeting of a strong non - oncogene addiction to dna - pkcs ( dna - dependent protein kinase catalytic subunit ) leads to the accumulation of dna double - strand breaks in atm - defective cancer cells, which triggers cell apoptosis via the proapoptotic signaling pathway [ 91 ]. these results are supported by our findings showing the dependence of myt1l - overexpressing glioblastoma cells on dna - pk. vx - 984 and m3814 are two selective dna - pk inhibitors [ 92, 93 ] that profoundly inhibit tumor growth in tumor xenograft models by enhancing radiotherapy. these two inhibitors are currently in ongoing clinical trials. the dna - pk / myt1l - cxcr1 - erk1 / 2 proliferative signaling loop we proposed here was primarily based on data obtained from glioblastoma cell lines. it is interesting to look at the expression of dna - pk, myt1l, and cxcr1 and the levels of phosphorylated erk1 / 2 in a large cohort of glioblastoma tissue samples and correlate the levels of these molecules to clinicopathological parameters of this disease. it is also interesting to look at the effect of myt1l on tumor growth in an in vivo tumor xenograft animal model using gfp - / myt1l - m059j and gfp - / myt1l m059k cells, further confirming our in vitro findings. western blot analysis the cells were rinsed twice with ice - cold pbs and scraped off the plate in radioimmunoprecipitation assay buffer ( ripa ). the total protein lysate prepared from human adult brain normal tissues was purchased from biochain ( newark, ca, usa ) and served as a normal control for human cell lines. the whole cellular lysates ( 30 – 100 µg per sample ) were electrophoresed via 10 % sds - page and electrophoretically transferred to pvdf membranes ( amersham hybondtm - p, ge healthcare, chicago, il, usa ) at 4 °c for 1. 5 h. the blots were incubated for 1 h with 5 % nonfat dry milk to block the nonspecific binding sites and subsequently incubated with polyclonal / monoclonal antibod ies specific to myt1l ( abnova, taipei, china ) or akt1 ( abcam, cambridge, gh, uk ), cdk2 ( abcam, cambridge, gh, uk ), dna - pkcs ( abcam, cambridge, gh, uk ), p21 ( abcam, cambridge, gh, uk ) or cdk4 ( cell signaling technology, danvers, ma, usa ), cdk6 ( cell signaling technology, danvers, ma, usa ), cyclin a2 ( cell signaling technology, danvers, ma, usa ), cyclin d1 ( cell signaling technology, danvers, ma, usa ), cyclin e1 ( cell signaling technology, danvers, ma, usa ), erk1 / 2 ( cell signaling technology, danvers, ma, usa ), p27 ( cell signaling technology, danvers, ma, usa ), perk1 / 2 ( cell signaling technology, danvers, ma, usa ) or bax ( santa cruz biotechnology, dallas, tx, usa ), bcl2 ( santa cruz biotechnology, dallas, tx, usa ), groα ( santa cruz biotechnology, dallas, tx, usa ), il8rb ( santa cruz biotechnology, dallas, tx, usa ), pakt1 / 2 / 3 ( santa cruz biotechnology, dallas, tx, usa ), or cxcr1 ( lsbio, lynnwood, wa, usa ) at 4 °c overnight. immunoreactivity was detected using a peroxidase - conjugated antibod y and visualized using the ecl plus western blotting detection system ( ge healthcare, chicago, il, usa ). the blots were stripped before reprobing with an antibod y against actin ( abcam, cambridge, gh, uk ) or gapdh ( santa cruz biotechnology, dallas, tx, usa ).