Cyclin-dependent kinases (CDK), as the family of serine-threonine kinases that transition from the G1 phase of the cell cycle to the S phase, are crucial for the control of eukaryotic cell proliferation. Cell cycle cyclins (CYC) are controlled by CDKs and cyclin-dependent kinase inhibitors (CDI). The levels of these proteins show differences at different phases of the cell cycle. Throughout the G1 phase, DNA replication is controlled. If DNA replication is appropriate, CDK is activated and the transition to the S phase starts with the creation of a positive feedback effect, which induces genome-wide transitional changes thereby triggering cell division.
CDK4 and CDK6 are the most studied CDKs, and are related to D-type cyclins (D1, D2, D3). Retinoblastoma (Rb), which is a tumor suppressor gene not phosphorylated under normal conditions before transition from the G1 phase to the S phase, is bound to members of the E2F transcription factor gene family. Cyclins acquire the phosphorylation property when the CDK complex is formed, and by phosphorylating Rb, which is a tumor suppressor gene, the E2F transcription factor is expressed and the progression of the cell cycle is obtained [11, 12,13]. If the CYC-CDK 4/6 complex is inhibited with CDI, the cell cycle is arrested in the G1 phase, the arrested cells age, and apoptosis develops. Therefore, interest has increased, and many studies have been conducted on this subject in many cancer types and their treatments.
As cancer is a pathological manifestation of uncontrolled cell proliferation, over the past decades many studies have examined the control point of cell proliferation. Thus it has been revealed that CYCs and CDKs are an effective strategy in the targeting of many cancer types and treatment . The increase in CDK 4/6 levels following endocrine treatment in hormone receptor positive (HR+) subtype of breast cancer and human epidermal growth factor receptor 2-negative (HER2-) advanced or metastatic breast cancer suggests that there may be a relationship between this pathway and resistance to endocrine treatment. Agents that show synergistic effects with anti-estrogen treatment have been developed with pre-clinical and subsequent clinical studies. The highly selective oral CDK4/6 inhibitors have been tested in combination with endocrine therapy in Phase III studies in metastatic breast cancer. The results have led to the US Food and Drug Administration approval of palbociclib (PD0332991) and ribociclib (LEE011), and abemaciclib (LY2835219) is in development. The indications for these drugs in the treatment of HR+/HER2- advanced breast cancer include use with an aromatase inhibitor (AI) as initial therapy in postmenopausal women and with Fulvestrant in women who showed disease progression during endocrine therapy.
Metabolic changes that emerge following CDK4/6 inhibition are related to irreversible inhibition of the cell cycle, cellular aging and apoptosis induction .
MCF-7 and BT474 breast cancer cell lines were used in this study as examples of luminal A and luminal B, which are the most common molecular subtypes (HR+/HER2-) of breast cancer. These two cell lines were treated with the highly selective CDK 4/6 inhibitors palbociclib and ribociclib to determine the potential resistance mechanisms of these new combination treatments by revealing potency and molecular differences despite CDK4/6 inhibition in the same pathway of the cell cycle, and the determination and comparison of cytotoxic effects and cell death pathways.
The results of the current study showed the dose and time-dependent cytotoxic efficacy of in vitro palbociclib and ribociclib as single agents in ER+/HER2-breast cancer cell lines. In the potential cell death pathways, different responses developed in the two drugs in the MCF-7 and BT474 cell lines. With a dose increase in ribociclib there was a proportional increase in the rate of DNA fragmentation, while in palbociclib there was observed to be an increase in the rate of DNA fragmentation up to a certain dose, and at higher doses this rate fell. This finding showed that as the increase in the rate of DNA fragmentation was dose dependent in ribociclib, the cytotoxic effect was related to apoptosis. Despite the cytotoxicity in increasing doses in palbociclib, the decrease that develops in the DNA fragmentation rate suggests that cell death pathways other than apoptosis play a role.
There are studies in the literature showing that of the cell death pathways, autophagy is used by palbociclib. In an in vitro study of stomach cancer cells by Brown et al, LC3B-2 levels were shown to be increased as a marker of autophagy as a result of treating the stomach cancer AGS cell line with palbociclib. It was also shown that there was a decrease in the p62/SQSTM1 level which is a protein fragmented by autophagy. Thus, it was shown that palbociclib induced autophagy . In a study of palbociclib by Capparelli et al, it was shown that CDK inhibitors induced autophagy and aging in fibroblasts related to cancer and there was increased expression of BNIP3, cathepsin B and ATG16L1, which are genes associated with autophagy .
Although there is limited information about cell death pathways induced by ribociclib, there are a few studies in the literature. To evaluate the apoptotic effect of ribociclib, HJ et al treated an aggressive thyroid cancer cell line (ATC) with ribociclib in an in vitro study, and using the immunoblot method showed that caspase-3-enzyme expression was increased, thereby demonstrating that ribociclib had an apoptotic effect .
Another mechanism in CDK4/6 inhibitor resistance is the activation of the P13K/AKT/mTOR pathway of breast cancer cells. mTOR is known to increase cell development and proliferation by suppressing autophagy . Michaloglou et al (2018) suggested that in ER+ cancer cell lines resistant to CDK 4/6 inhibitors, sensitivity to CDK 4/6 inhibitors is regained by mTORC1/2 inhibition .
When other cell death pathways are considered, various studies are showing that there is a relationship between palbociclib and autophagy. There are various opinions on this issue. The results of the current study show that palbociclib inhibits the cell cycle through autophagy. Upon seeing that palbociclib uses the autophagy route in various studies, we set up an autophagy experiment considering that the reason for the decrease in apoptosis at 100 micromolar dose of palbociclib in our apoptosis experiment was due to palbociclib's use of the autophagy pathway. In this autophagy experiment, we found that palbociclib uses the autophagy pathway, but ribociclib does not use the autophagy pathway even at the highest doses. Both of these two CDK 4/6 inhibitors were found to have increased cytotoxicity depending on dose and time. Ribociclib, one of the CDK 4/6 inhibitors, only used the apoptosis pathway, while the other CDK 4/6 inhibitor, palbociclib, used the apoptosis pathway to a certain dose. However, with increasing doses, autophagy replaced apoptosis. The use of autophagy inhibitors in the treatment, especially in patients receiving palbociclib, suggests that it may have a positive effect on survival in the treatment of resistance to CDK 4/6 inhibition. At the same time, it should be kept in mind that this difference of both drugs may play a role in the differences that may develop in terms of side effect profiles. Considering the data in the literature, this study is the first study to compare the cell death pathways of drugs in breast cancer and should be supported by in-vivo studies.