Cell cycle is defined as the process through which the cell replicates all its genomic material and divides into two identical cells (Alberts et al., 2002). It consists of four phases: gap 1 (G1), where the cell grows in size and transcribes the RNA and protein necessary during cell division; synthesis or S phase, where all chromosomes are being replicated; gap 2 (G2), where cell growth and protein synthesis continue; and mitosis or M phase, where the cell restructures its membrane and organizes the newly synthesized chromosomes and then divides into two daughter cells. Before entering cell cycle, highly proliferative cells such as stem cells and lymphocytes are in a reversible cell cycle arrest, known as quiescence or gap 0 (G0). However, other cells such as neurons or adipocytes are irreversibly arrested in G0 phase, a phenomenon often described as cellular senescence. Senescence is also predominant in highly damaged cells, acting as a protective mechanism during the DNA damage response (DDR) (Terzi et al., 2016).

Each cell cycle phase, as well as transitions from one phase to the other, are tightly regulated by interactions between cyclins and cyclin-dependent kinases (CDKs) (Johnson and Walker, 1999). In general, cyclins directly bind CDKs and induce the formation of cyclin—CDK complexes. This promotes CDK activity and therefore ensures activation of specific transcriptional programs that allow cell cycle progression. More than ten CDKs are known to be involved in various events during cell cycle. From these, CDK1, 2, 3, 4, and 6 directly mediate cell cycle progression.

Transition from quiescence or G0 phase in G1 phase is modulated by growth factor signals or mitogenic stimulation. These result in the upregulation of Cyclin D, which binds to and activates CDK4 and CDK6 to promote cell commitment to enter G1 phase (Jinno et al., 1999; Lea et al., 2003). High CDK4/6 expression and activation ensures cell progression through G1 phase (Mende et al., 2015; Topacio et al., 2019).

On the molecular level, CDK4 and 6 phosphorylate Retinoblastoma (Rb) and promote the accumulation of E2F, a direct regulator of genes necessary during DNA synthesis. Furthermore, CDK4 and CDK6 activation initiates cell growth through activation of mammalian target of rapamycin complex 1 (mTORC1) (Romero-Pozuelo et al., 2020). Besides, CDK4 and 6 are involved in the control of DNA replication mechanisms (Braden et al., 2008). Along with CDK4/6, CDK2 and CDK3 are also activated during G1 phase. Rb phosphorylation, and therefore the accumulation of E2F during G1 phase, directly mediate the upregulation of Cyclin E in late G1 phase, which binds and activates CDK2. Formation of CDK2/Cyclin E complex maintains Rb phosphorylated in order to promote G1/S phase transition (Massague 2004; Horiuchi et al., 2012). However, CDK3 upregulation during late G1 phase seems to be independent of Cyclin D, E or A binding (Braun et al., 1998). Interestingly, the upregulation of CDK2 has been also shown to be important during the G1/S checkpoint in response to DNA damage. For example, knocking-down CDK2 in the HCT116 tumor cell line significantly reduced p53 phosphorylation in response to hydroxyurea (HU) and suppressed G1/S cell cycle arrest (Bacevic et al., 2017). Some recent studies also described a role of CDK2 directly after mitosis, as an intermediate level will remain in the cells that continue proliferating, while those that lack CDK2 can enter quiescence or so called gap 0 (G0) (Spencer et al., 2013; Gookin et al., 2017). On the other hand, high levels of Cyclin C/CDK3 have been reported to directly mediate quiescence (Ren and Rollins 2004).

The beginning of S phase is marked by increasing levels of Cyclin A, which binds CDK2. The complex formed by Cyclin A/CDK2 drives the cells through S phase and promotes DNA replication. During late S/G2 phase, increased levels of Cyclin A induce CDK1 activation, which drives entry into mitosis (Gavet and Pines, 2010; Kalous et al., 2020). Later, the formation of CDK1/Cyclin B complex triggers progression through M phase. Along with its important role in successful cell mitosis (Vassilev et al., 2006), CDK1 can also influence the remodeling of cell adhesion complexes during G1, S and G2 cell cycle phases (Jones et al., 2018) and promotes protein synthesis during proliferation (Haneke et al., 2020). Interestingly, CDK1 is reported to be the only necessary cyclin-dependent kinase during cell cycle, being able to bind to all cyclins and drive all events during cell division (Santamaria et al., 2007).

Several other CDKs are known to be involved in cell cycle progression as well. CDK7, for example, is an important cell cycle regulator. Its binding to Cyclin H and mating-type 1 protein (Mat1) induces the formation of CDK-activating kinase (CAK) complex. CAK activity is crucial to promote CDK2 and CDK1 binding to cyclins, therefore allowing cell division (Fisher and Morgan, 1994; Larochelle et al., 2007; Olson et al., 2019). CDK5 upregulation is mostly observed in, but not limited to, neurons, and is often correlated to cell apoptosis. Nevertheless, it can also regulate the cell cycle by phosphorylating Rb and interacting with E2F during G1 phase (Zhang et al., 2010; Chang et al., 2012; Futatsugi et al., 2012). CDK8 is a partner of Cyclin C and its expression has been shown to be important in stabilizing Cyclin C activity during cell cycle (Tassan et al., 1995; Barette et al., 2001). Interestingly, CDK8 and Cyclin C, as well as CDK19/Cyclin C complex, are strongly required during p53-dependent p21 transcriptional activation, for cell cycle arrest in response to DNA damage (Donner et al., 2007; Audetat et al., 2017). Last, cyclin-dependent kinases such as CDK9 and CDK13 are not directly controlling cell cycle phase transitions, but are rather involved in transcription mechanisms, by associating with Cyclin T or Cyclin K (Garriga et al., 2003; Yu et al., 2010; Greifenberg et al., 2016).

To summarize, entry into cell cycle depends on mitogenic or growth factor signals. CDK4/6/Cyclin D complex formation promotes Rb phosphorylation and accumulation of free E2F, which ensures progression through G1 phase. CDK5 activity also increases E2F levels during G1. High levels of E2F during late G1 induce CDK2/Cyclin E complex that in return further phosphorylates Rb and promotes G1/S transition. At the beginning of S phase, Cyclin E levels decrease and CDK2 forms a complex with the increasing Cyclin A, which not only ensures progression through S phase, but also transition into G2 phase. CDK2/Cyclin A complex is especially regulated by the CDK7/Cyclin H/Mat1 complex, also described as CAK. CAK also regulates CDK1/Cyclin A complex formation during late G2 and Cyclin B binding to CDK1 during mitosis. Any disturbances to the cell cycle machinery will result in cell cycle arrest. CDK2 and CDK3 are especially important in mediating either quiescence or senescence. Indirectly, CDK8, 9, 13, and 19 also mediate cell cycle, being involved in the transcription machinery, while CDK5 can directly modulate apoptosis as well. A schematic representation of the important role of CDKs in cell cycle is shown in Figure 1. While normal cells are able to activate the necessary mechanisms for cell cycle arrest when the DNA is damaged, these pathways are usually suppressed or non-existent in tumor cells, enabling them to continue progression through cell cycle. The following sections will address the CDK’s role in the tumor cell division and how therapies targeting CDKs can modulate CRC development.

Changes in the regulatory mechanisms that control cell division are often related to accumulation of mutations and/or epigenetic dysregulations of cancer related genes and can contribute to the molecular mechanisms of colorectal cancer (CRC). CRC tissue often shows changes in genes related to cell cycle arrest (p16 and p21), apoptosis (p53) or proliferation (PCNA) (Yue et al., 2003; Kruschewski et al., 2011). Multiple other mutations have also been described to be involved in CRC development. As a result, CDKs expression can be changed in tumor cells.

When looking at the signature of differentially expressed genes (DEGs) in patients with CRC compared to normal colon tissue, an upregulation in CDK1 gene expression is often observed (Zhao et al., 2019; Ding et al., 2020; Li et al., 2020). Interestingly, the expression of CDK1 in the nucleus and cytoplasm has been used as a marker to describe patterns in the overall survival of patients with CRC (Sung et al., 2014). Staining of over 164 cancer samples from primary CRC revealed that CDK1 is expressed in both cell nucleus and cytoplasm to a certain degree. The evaluation of nuclear/cytoplasm (N/C) ratio on these samples showed that high N/C expression is often found in patients with overall worse survival and a N/C > 1.5 can be considered a risk factor. Furthermore, high CDK1 expression is predominant in patients with resistance to 5-fluorouracil (5-Fu), a common CRC treatment, and it seems to reduce the effect of chemotherapy (Zhu et al., 2020). An upregulation of CDK1 in CRC has been also observed in response to other drugs such as: betaxol, penbutolol and propofol amongst others (Mastrogamvraki and Zaravinos, 2020).

CDK2, 4 and 6 levels in CRC are closely related to the Rb protein hyperphosphorylation, which seems to promote cancer progression. CDK4/6 is usually amplified in colon tumors compared to healthy epithelium (Mastrogamvraki and Zaravinos, 2020; Jardim et al., 2021). Abundant levels of CDK4 are especially observed in CRC patients with enhanced dysplasia and are correlated to increased tumor cell proliferation (Zhang et al., 1997; Bartkova et al., 2001). Some CDK2 expression is normally found in healthy epithelium. However, its upregulation can be predominantly observed human CRC tissue samples (Yamamoto et al., 1995). Interestingly, CDK2 overexpression in primary CRC tumors is also linked to lymph nodes metastasis, but not liver metastasis (Li et al., 2001; McCurdy et al., 2017). Nevertheless, a certain CDK2 activity has been reported to improve recurrence-free survival (RFS) of patients after surgery (Yamamoto et al., 1995). A similar pattern to CDK2 expression in CRC is observed in CDK3 levels as well. Its overexpression has been linked to metastasis and tumor cell invasion, where it seems to be promoting epithelial to mesenchymal transitions (Lu et al., 2016).

CDK5 expression is also reported to be much higher in CRC cells compared to normal epithelium and it correlates to increased tumor growth and poor prognosis (Zhuang et al., 2016; de Porras et al., 2019). Most important, CDK5 is directly involved in the degradation of the cell cycle inhibitor p21 and can enhance CDK2 activity, which might further promote tumor cell growth (Huang et al., 2016). Decreased survival rates are also observed in CRC patients with high CDK9 and CDK13 levels (Kim et al., 2012; Wang et al., 2019). Interestingly, high CDK9 expression in CRC tissue was negatively correlated with cytotoxic CD8⁺ T cell infiltration. Furthermore, these infiltrated cells showed increased cell exhaustion in CDK9-high tumors, which might further affect patient outcome (Wang et al., 2019). Last, CDK8 overexpression in CRC is also considered as a marker for poor patient prognosis, being directly linked to β-catenin activation amongst others and therefore promoting cancer growth (Firestein et al., 2008; Firestein et al., 2010; Seo et al., 2010). Overall, cyclin-dependent kinase activation is often observed in colorectal cancer and seems to promote tumor progression and an overall worse survival of patients, as summarized in Table 1.

Basic research using murine knockout models or in vitro gene silencing in tumor colon cancer cell lines also provided some understanding for the relevance on CDKs in CRC development. Since CDKs are vital components of the cell cycle, creating knockout mouse models is usually unsuccessful. This is because most CDKs (e.g. CDK1, 4, 6, 9, and 13) are critical during embryonic development, as is summarized in (Campbell et al., 2020). Similarly, conditional knockout models often show severe impairments.

Nevertheless, some fundamental research data in regards to the role of CDKs in colorectal cancer are available. For example, it is know that CDK4 activation in CDK4^R24C/R24CApc^+/min mice leads to significant increased in tumor vascularity in comparison to CDK4^+/+Apc^+/min mice or APC^+/min mice (Abedin et al., 2010), while knocking out CDK4 in APC^+/min mice reduces adenoma development (Karim et al., 2013). CDK5 silencing via transfection can directly reduce the proliferation of human HCT116 and SW480 tumor cell lines (Zhuang et al., 2016). Similarly, knocking down CDK9 in HCT116 and HT29 tumor cell lines induced their apoptosis by Caspase 7 cleavage (Rahaman et al., 2019). Furthermore, it reduced Cyclin D1 protein expression, suggesting cell cycle arrest induction in these cells.

Stable silencing of CDK8 and CDK19 in Colo205 human colon cancer cells reduced β-catenin/TCF-dependent transcription (Dale et al., 2015). A direct link between CDK8 and β-catenin regulation in tumor cell proliferation and death has also been described, where inactivation of CDK8 by siRNA transfection in HCT116 cells significantly reduced the RNA and protein levels of β-catenin (He et al., 2011). Generally, silencing CDK genes in colon cancer cells reduces their proliferation and induces cell death, which makes them an attractive target for the development of new inhibitory therapies.

CDK inhibitors are also often used in basic research to understand molecular mechanisms of CDK activation in cell cycle regulation or tumor cell proliferation. This section describes the current understanding on the potential use of various CDK inhibitors to mediate colorectal cancer development.

When thinking about preventing cell cycle progression and proliferation of tumor cells, CDK4/6 inhibitors are very efficient. The most commonly used are Ribociclib, Palbociclib, Abemaciclib and Trilaciclib. CDK4/6 inhibitors are especially effective at treating breast cancer amongst others, many of them being nowadays tested in phase I and II clinical trials (Wu et al., 2020). Nevertheless, they are also being tested as therapy for colorectal cancer. A schematic representation of the mechanism of action of CDK4/6 inhibitors is shown in Figure 2.

There is no doubt that targeting cell cycle machinery, and especially cyclin-dependent kinase activity of tumor cells, offers new opportunities to treat patients with advanced colorectal cancer. Nevertheless, cancer itself is a multifactorial disease and therefore the treatment with just one drug is not always successful.

CDK4/6 inhibitor therapy in particular shows promising results in the relief and stabilization of the patients, but its effect is amplified when used in combination with other treatments. More recent studies have focused on evaluating therapeutic potential of CDK4/6 inhibitors when coupled with other drugs in treating CRC. For example, when treating tumors in patient-derived Rb+ colorectal xenograph models, the authors found that a combination of MEK inhibitor Trametinib with Palbociclib significantly reduces tumor volume in comparison to monotherapy. Furthermore, KRAS-mutated cells were especially sensitive to this treatment (Lee et al., 2016; Ziemke et al., 2016). Similiar results were obtained when using a Raf inhibitor (LY3009120) in combination with Abemaciclib, where Ras- and Braf-mutated CRC was especially sensitive to this treatment (Chen et al., 2018). Last, the combination of checkpoint inhibitors like anti-PD1 therapy (SHR-1210) with CDK4/6 inhibitor (SHR6390) is currently evaluated in Phase I and II clinical trial for advanced colorectal cancer (ClinicalTrials.gov Identifier: NCT03601598), but no data have been published yet.

Further studies are necessary for understanding the potential of targeting CDK4/6, together with other genes involved in cell cycle machinery. For example, tumor cells depend on high telomerase activity, which enables them to preserve the telomeres during extensive proliferation. Inducing telomere dysfunctions in tumor cells, using the telomere-specific inhibitor 6-thio-dG, potentiates antitumor responses in mice bearing MC38 tumors (Mender et al., 2020). Therefore, combining CDK4/6 inhibitors for cell cycle arrest and 6-thio-dG might provide a more efficient tumor targeted therapy.

One significant challenge raised by the use of CDK4/6 inhibitors is its effect on normal cells, and especially on the highly proliferating cells, such as activated immune cells found in the tumor microenvironment. Targeting CDKs might disrupt the function of upstream genes involved in the cell cycle, such as sirtuins, in normal cells. Modifications in sirtuin 1 (SIRT1) function are especially important. Even though SIRT1 is also upregulated in the CRC tissue compared to the normal one and it has been linked to tumor size and invasion (Chen et al., 2014; Yu et al., 2016), its function in haematopoiesis is nevertheless crucial (Rimmele et al., 2012). Dysfunctions in SIRT1 in normal cells due to CDK4/6 inhibitor use might therefore potentiate cellular senescence and premature aging in various cellular compartments (Sasaki et al., 2006).

Overall, CDK inhibitors are efficient in preventing colon tumor cells from proliferating by inducing cell cycle arrests, and, in some cases, even apoptosis, making them useful for developing new potential therapeutic strategies for CRC. Nevertheless, a comprehensive analysis on how CDK inhibitors might affect normal cells, as well as the antitumor response of immune cells to CRC, would enhance our understanding on this novel therapy.