**2. DNA Damage Checkpoint Promotes DNA Damage Recognition and Cell Cycle Arrest: The First Barrier to Cancer Progression**

When DNA damage arises, a complex network of signaling cascades named DNA damage checkpoint (DDC) are activated to recognize DNA damage and arrest the cell cycle, thus ensuring enough time to cells to repair the lesion or eventually leading to senescence/apoptosis. These processes are tightly regulated to guarantee cell survival by promoting genomic stability and reducing the possibility that lesions are inherited, after cell division. Similar to other cellular signaling cascades, DDC is driven by protein phosphorylation and at its core there are three upstream kinases: ATM, ATR, and DNA-PKs [15] (Figure 1); moreover, recent evidence also suggests that non-coding (nc)RNA molecules and the RNA interference (RNAi) factors Drosha and Dicer have a role in this mechanism [16,17].

Each kinase is recruited to DNA lesions by specific co-factors: ATM is recruited by the C terminus of NBS1 a component of the MRE11-RAD50-NBS1 (MRN) complex, DNA-PKs is recruited by Ku80/Ku70 heterodimer and ATR is recruited by its stable binding partner ATRIP-RPA. In general, the first two kinases are involved in the initial recognition of DSBs, then, ATM promotes either HR or NHEJ, while DNA-PK activity is restricted to NHEJ. Instead, ATR is recruited to all kind of lesions that generate ssDNA coated by replication protein A (RPA) such as: DSBs after end resection, bulky lesions when the damaged strand is excised by NER and stalled replication forks when the double helix is opened and/or processed by nucleases. ATR is also involved in the recognition of particular DNA damage such as UV-damaged DNA [18]. Following DNA damage recognition, all the three

kinases determine cell cycle arrest by interacting with specific substrates (Figure 1). ATR and ATM phosphorylate and activate the tumor suppressor checkpoint kinase 1 (CHK1). CHK1 regulates the G2/M checkpoint by activating kinase WEE1, which in turn phosphorylates the Cyclin-dependent kinase 1 (CDK1) reducing its activity and preventing entry into mitosis; CHK1 also regulates S phase checkpoint by promoting CDC25A phosphatase degradation, whose activity is essential to remove inhibitory phosphate groups from kinases CDK4 and CDK2 and guarantee cell cycle progression [19]. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 30

**Figure 1.** DDC signaling cascade determines arrest in specific phases of the cell cycle. ATM, DNA-PK, and ATR phosphorylate the downstream kinases CHK2 and CHK1; in turn they activate P53 and WEE1 that, by inhibiting CDK6/4 and CDK1 determine arrest in G1 and G2/S, respectively. CHK1 and CHK2 also inhibit CDC25 phosphatase that does not remove inhibitory phosphate groups from CDK2/4, therefore determining arrest in the S phase. **Figure 1.** DDC signaling cascade determines arrest in specific phases of the cell cycle. ATM, DNA-PK, and ATR phosphorylate the downstream kinases CHK2 and CHK1; in turn they activate P53 and WEE1 that, by inhibiting CDK6/4 and CDK1 determine arrest in G1 and G2/S, respectively. CHK1 and CHK2 also inhibit CDC25 phosphatase that does not remove inhibitory phosphate groups from CDK2/4, therefore determining arrest in the S phase.

Each kinase is recruited to DNA lesions by specific co-factors: ATM is recruited by the C terminus of NBS1 a component of the MRE11-RAD50-NBS1 (MRN) complex, DNA-PKs is recruited by Ku80/Ku70 heterodimer and ATR is recruited by its stable binding partner ATRIP-RPA. In general, the first two kinases are involved in the initial recognition of DSBs, then, ATM promotes Due to its activity among replication fork collapse, ATR-CHK1 axis protects cells from replication stress and mitotic catastrophe that could arise after uncontrolled division [20]. ATM and DNA-PK also phosphorylate the tumor suppressor checkpoint kinase 2 (CHK2); CHK2 stabilizes P53, which inhibits CDK6 and CDK4 leading to cell cycle arrest in G1/S phase [21].

either HR or NHEJ, while DNA-PK activity is restricted to NHEJ. Instead, ATR is recruited to all kind of lesions that generate ssDNA coated by replication protein A (RPA) such as: DSBs after end resection, bulky lesions when the damaged strand is excised by NER and stalled replication forks when the double helix is opened and/or processed by nucleases. ATR is also involved in the recognition of particular DNA damage such as UV-damaged DNA [18]. Following DNA damage recognition, all the three kinases determine cell cycle arrest by interacting with specific substrates Thanks to its inhibition of cell cycle progression, the activation of DDC is considered the first barrier to tumor progression and the loss of one or more DDC-involved proteins determines accumulation of un-repaired DNA lesions which greatly contribute to cancer initiation and progression. Indeed, DDC helps cells recover from DNA injuries and overexpression of its players is frequent in cancer resistant to DNA damaging agents.

degradation, whose activity is essential to remove inhibitory phosphate groups from kinases CDK4

Due to its activity among replication fork collapse, ATR-CHK1 axis protects cells from replication stress and mitotic catastrophe that could arise after uncontrolled division [20]. ATM and

and CDK2 and guarantee cell cycle progression [19].

(Figure 1). ATR and ATM phosphorylate and activate the tumor suppressor checkpoint kinase 1

ATM is a well-recognized tumor suppressor and its alterations are common in several cancers including breast, gastric, colorectal, and prostate cancer [22]. Mutations in DNA-PK have been found associated to several cancers such as prostate cancer and melanoma [23,24]. Finally, ATR mutations promote development of melanoma and oropharyngeal cancer syndrome [25,26].

After initial recognition of DNA damage and cell cycle arrest, cells undergo specific DNA repair pathway depending on the cell cycle phase and type of damage.
