Pan-cancer antagonistic inhibition pattern of ATM-driven G2/M checkpoint pathway vs other DNA repair pathways

Cancer is one of the leading healthcare burdens with nearly 10 million deaths occurred globally in 2020 [1]. The number of cancer patients is estimated to be 28.4 million by 2040, which gives a figure of 47% rise from 2020 [1]. The carcinogenesis is strongly associated with DNA mutations and loss of genome integrity which leads to altered gene expression and occurrence of oncogenic gene products. For example, genetic and epigenetic alterations lead to cancer development through activation of proto-oncogenes and inhibition of tumor suppressor genes [2]. A deficiency of DNA repair which leads to extreme accumulation of genetic changes in cell is considered one of the major molecular mechanisms of cancer development [3]. For example, the tumor suppressor gene for ATM kinase which plays an important role in the response to DNA damage is mutated in ~30–50% of mantle cell lymphomas [4]. Inactivating gene mutations in its partner kinases ATR and DNA-PK (catalytic subunit of DNA-dependent protein kinase) can also lead to cancer [5].

Many tumor types have strong genetic and gene expression intragroup heterogeneities, as demonstrated in several global projects assessing cancer genome, transcriptome, methylome and proteome data at the large scale, such as The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) [6]. However, DNA repair may play a dual role in carcinogenesis: by keeping DNA integrity it limits tumor-associated alterations and molecular evolution, but on the other hand it can protect the cell from DNA damage, e.g. by promoting tumor survival following radiation therapy and genotoxic chemotherapy [7]. Indeed, some types of anticancer therapies such as ionizing radiation and chemotherapies can directly damage DNA or prevent its successful biosynthesis during mitosis, which can lead to cancer cell death unless timely and properly repaired [8]. Thus, understanding the changes in regulation of DNA repair pathways in different tumors can strengthen current and future therapeutic approaches.

There are several types of DNA repair pathways which execute different functions [9]. For example, DNA base damage is corrected through the base excision repair (BER) mechanism or by direct reversal of DNA lesion. Multiple and bulky base damage is neutralized by nucleotide excision repair (NER) mechanism including global genome repair (GGR) and transcription-coupled repair (TCR) mechanisms, mismatch repair (MMR), interstrand cross-link repair (ICL, Fanconi anemia pathway), and translesion synthesis. DNA breaks can be restored by single or double strand break repair systems through the homologous recombination (HR) and non-homologous end joining (NHEJ) mechanisms [9], [10].

DNA repair pathways also deal with DNA damage checkpoint signaling cascades which recognize DNA lesions and cause arrest of the cell cycle, thus allowing cells to repair damage or activate senescence/apoptosis processes [11], [12]. Such checkpoints control the G1/S, S, and G2/M phase transitions, and thereby integrate DNA repair processes into the cell cycle [12], [13]. The checkpoint mechanisms are mainly based on the control of phosphorylation by three kinases: ataxia-telangiectasia mutated (ATM), ATM and RAD3-related (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PK). Each of them is involved in DNA damage recognition and DNA repair activation [14], [15], [16]. For example, ATR and ATM phosphorylate and activate checkpoint kinase 1 (CHK1) [17], [18], [19]. CHK1 regulates the G2/M checkpoint by activating the WEE1 kinase, which phosphorylates cyclin-dependent kinase 1 (CDK1), thus reducing its activity and preventing entry into mitosis in response to DNA damage [12].

Many individual DNA repair actors and their molecular ensembles have been investigated in detail in health and disease using available genomic, transcriptomic, and proteomic data [20], [21], [22]. Recently, using publicly available TCGA cancer RNA expression data we detected an overall pan-cancer increase in the activation levels of the DNA repair pathways compared to an overall inhibition of the activities of the signaling, cytoskeleton or metabolic pathways [23]. However, deeper investigation of thyroid cancer profiles revealed that although most of the DNA repair pathways were indeed hyperactivated, the G2M checkpoint pathway was in contrast inhibited in the cancer tissues [24]. This remained unclear, however, whether this trend exists also in the different cancer types. We, therefore, aimed to investigate in detail the profiles of DNA repair pathway activation in major solid cancers using the literature and our experimental RNA sequencing data which could cross-validate each other. Based on the public databases, we also compared the RNA- and proteome-level results.

DNA repair mechanisms keep genome integrity and limit tumor-associated alterations and heterogeneity, but on the other hand they promote tumor survival after radiation and genotoxic chemotherapies. We screened pathway activation levels of 38 DNA repair pathways in nine human cancer types (gliomas, breast, colorectal, lung, thyroid, cervical, kidney, gastric, and pancreatic cancers). We took RNAseq profiles of the experimental 51 normal and 408 tumor samples, and from TCGA/CPTAC project databases - of 500/407 normal and 5752/646 tumor samples, and also 573 normal and 984 tumor proteomic profiles from Proteomic Data Commons (PDC) portal. For all the samplings we observed a congruent trend that all cancer types showed inhibition of G2/M arrest checkpoint pathway compared to the normal samples, and relatively low activities of p53-mediated pathways. In contrast, other DNA repair pathways were upregulated in most of the cancer types. The G2/M checkpoint pathway was statistically significantly downregulated compared to the other DNA repair pathways, and this inhibition was strongly impacted by antagonistic regulation of (i) promitotic genes CCNB and CDK1, and (ii) GADD45 genes promoting G2/M arrest. At the DNA level, we found that ATM, TP53, and CDKN1A genes accumulated loss of function mutations, and cyclin B complex genes – transforming mutations. These findings suggest importance of activation for most of DNA repair pathways in cancer progression, with remarkable exceptions of G2/M checkpoint and p53-related pathways which are downregulated and neutrally activated, respectively.

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