Disturbances in HDACs expression is implicated as a possible carcinogenesis trigger, due to its vital role in controlling the expression of many oncogenes and tumor suppressor genes as well as its involvement in controlling the activity of several transcriptional factors. (Seto andYoshida, 2014). Uncontrolled cell proliferation is one of the hallmarks of cancer and one of its triggers might be through nuclear and cytoplasmic disturbances in HDAC expression (Sharma et al., 2010).
That’s why Epigenetic modulators such as HDACs inhibitors have been tested in many studies as potential anti-cancer drugs. Indeed to date, the FDA has approved three HDAC inhibitors; vorinostate, romidepsin, and belinostat for cutaneous/peripheral T-cell lymphoma treatment (Mottamal et al., 2015). Sodium butyrate (NaBu), a short chain fatty acids which acts as class I/IIa HDAC inhibitor, is under clinical trials as anti-cancer agent against various types of cancer (Davie, 2003). The reported alteration in HDACs expression in MDA-MB-231(Kalle andWang, 2019) prompted us to investigate the possible modulatory effect of NaBu on abemaciclib anti-cancer activity. Abemaciclib is FDA approved CDK4/6 inhibitor and it is used for advanced metastatic hormonal positive breast cancer alone or in combination with endocrine therapy (O'Brien et al., 2018).
In the present study, MDA-MB-231 cells were found to show an overexpression of cyclin D1, and upon treating the cells with abemaciclib for 48h, it failed to reduce cyclin D1 protein level. This result is consistent with a previous study where abemaciclib failed to decrease cyclin D1 expression in colorectal cancer. (Lee et al., 2020) Abemaciclib also showed an elevation in the hypophosphorylated form of Rb tumor suppressor protein in the present study. Consistently, it was reported that abemaciclib treatment caused accumulation of cyclin D1 together with a partial inhibition in Rb phosphorylation in HCT116 colorectal cancer cell line (S. H. Chen et al., 2018). It is well recognized that abemaciclib through its inhibitory effect on CDK4/6 results in inhibition of Rb phosphorylation which restricts the cell cycle progression. (O'Brien et al., 2018).To confirm the implication of cyclin D1 accumulation in the partial inhibition of Rb phosphorylation, cyclinD1 knockout cells were treated with abemaciclib, and a complete inhibition in Rb phosphorylation was noted in colorectal cancer cells. (S. H. Chen et al., 2018)
In the present study, NaBu was able to reduce cyclin D1 protein level as well as elevate the hypophosphorylated form of Rb. This is consistent with the previously reported decrease in cyclin D1 level upon treatment of monoplastic leukemia cell line (SKM-1) and non-small lung cancer cell line (NCI-H23) with NaBu. (M. Huang et al., 2004; Pellizzaro et al., 2001) The ability of NaBu to decrease cyclinD1 expression might be due to its inhibitory effect on NF-κB, which is considered as a direct transcription activator of cyclinD1. (Hu andColburn, 2005) NaBu could also inhibit cyclin D1 expression through inhibition of the cytoplasmic HDAC6. (Kalle andWang, 2019) Briefly, inhibition of HDAC6, leads to hyperacetylation and proteosomal degradation of HSP90 which in turn leads to unstabilization and degradation of EGFR, thus attenuating all mitogenic signaling pathways downstream of EGFR activation, such as MAPK/ERK1-2 and PI3K/AKT/mTOR signaling pathways (Wee andWang, 2017). Inhibition of MAPK/ERK1-2 pathway attenuates the formation of AP-1 transcription factor which is a direct activator of cyclinD1 expression. In addition, inhibition of PI3K/AKT/mTOR signaling pathway activates GSK-3β and that leads to phosphorylation and proteosomal degradation of cyclinD1 protein (Ghoneum andSaid, 2019; Guo et al., 2011; Wee andWang, 2017).
Various possible mechanisms could be linked to the effect of NaBu on Rb phosphorylation. First, NaBu is able to induce p53 wild type expression (J. Chen et al., 2019), this was confirmed in the present study, as NaBu elevated both the gene and protein level of p53. As p53 is a stress detector transcription factor, so its expression is elevated during DNA damage and nutrient deficient or any sort of cellular stresses such as that induced by drugs or free radicals. The expression of p53 controls the cell fate, as one of its roles is to directly induce the transcription of p21 and p27, which are endogenous CDKs/cyclins complexes inhibitors, with a consequent attenuation of Rb phosphorylation level, to stop the cell cycle progression and give much more time to the cell to repair the DNA damage or to restore its energy in case of nutrient deficient (Vermeulen et al., 2003). Thus the ability of NaBu to induce p53 expression could be linked to its ability to induce a cell cycle arrest as was noted in HCT116 colon cancer treated with NaBu (J. Chen et al., 2006).
It is worth mentioning that NaBu could activate p21 and p27 transcription through inhibition of PI3K/AKT signaling pathway secondary to inhibition of HDAC6 as mentioned before (Ghoneum andSaid, 2019; Wee andWang, 2017). Another possible mechanism for diminished Rb phosphorylation by NaBu treatment could be mediated through inhibition of nuclear HDAC1 which is an inhibitor of SP-1 transcription factor. Activation of SP-1 could also induces the transcription of p21. (Pajak et al., 2007) In addition, NaBu was reported to decrease c-myc oncogene expression which acts as a transcription repressor of p21.(J. Chen et al., 2019; La Monica et al., 2020)
In the present study the combination of NaBu and abemaciclib enhanced the ability of abemaciclib to induce a cell cycle arrest through decreasing cyclinD1 expression with a further elevation in hypophosphorylated Rb level and the effect was maintained in small doses combination.
Abemaciclib and NaBu as single drugs were able to reduce expression of the cell cycle driver E2F2 in the current study. This decrease could be mediated through their ability to reduce c-myc expression which is a direct activator of E2F2 expression. (Beier et al., 2000) Previously, a significant decline in c-myc level upon treatment of mutant non-small lung cancer cells with abemaciclib or NaBu treatment was noted (La Monica et al., 2020). It was also reported that the degradation of HSP90 induced by NaBu causes unstabilization and degradation of E2F1,E2F2 and E2F3 (Kotwal et al., 2021).
Abemaciclib and NaBu as a single drugs attenuated CDK2 gene and protein level. Consist with this finding, it was reported that abemaciclib reduced CDK2 expression in a various cancer cells and it was considered as a pharmacodynamic biomarker, indicating the responsiveness of the patient to the treatment (O'Brien et al., 2018). A significant decrease in CDK2 expression upon NaBu treatment was also previously noted in monoplastic leukemia cells (M. Huang et al., 2004). This decrease could be due to the observed decrease in NF-κB level which acts as a direct transcription activator of CDK2 gene, as well as indirect activator through increasing E2F3 level. (Ledoux andPerkins, 2014).
Recently, a link between CDK6 inhibition and NF-κB phosphorylation, particularly P65 subunit was noted in cervical cancer cell line. CDK6 phosphorylates p65 subunit and ease its translocation to the nucleus leading to the transcriptions of genes implicated in cell proliferation, angiogenesis, metastasis and resisting apoptosis (Buss et al., 2012).Thus, inhibition of CDK6 by abemaciclib may lead to attenuation of NF-κB transcription activity and its expression.
On the other hand, acetylation of p65 subunit could activate or repress p65 transcription activity according to the site of acetyl group. HDAC3 has the ability to remove the acetyl group from the inhibitory residues on p65 subunit. Thus inhibition of HDAC3 by NaBu could attenuate p65 transcription activity and in turns decrease NF-κB level (Leus et al., 2016).
Abemaciclib reduced AKT phosphorylation level in the present study which goes in parallel with abemaciclib-induced decrease in non-small lung cancer cell line (La Monica et al., 2020) In contrast to other CDK4/6 inhibitors such as palbociclib which showed elevation in AKT phosphorylation level in MDA-MB-231 breast cancer cell line. On the other hand, NaBu had no effect on AKT phosphorylation as a single drug despite its ability to attenuate EGFR signaling pathway (W. Huang et al., 2019). A similar observation was noted in cervical cancer cell line (J. Chen et al., 2006).
By analyzing the cell cycle regulators, abemaciclib significantly elevated p53 gene expression but it failed to stabilize its protein level. Similarly, abemaciclib failed to induce p53 level in liposarcoma cell line (Sriraman et al., 2018). In various cancer types, abemaciclib failed to induce apoptosis and its effect was restricted to inhibition of cell proliferation (La Monica et al., 2020; Lee et al., 2020). On the other hand, NaBu showed a significant elevation in p53 gene and protein level. This finding is in line with the reported induction of intrinsic apoptosis, through p53 dependent pathways, in MCF7 breast cancer cell line and HCT116 colon cancer cell line following butyrate treatment (J. Chen et al., 2019). The mechanism by which NaBu stabilized p53 protein level might be through its inhibitory effect on HDAC1. Normally, HDAC1 deacetylates p53 protein leading to recruitment of MDM2 E3 ubiquitin ligase, and proteosomal degradation of p53. (Seto andYoshida, 2014) Stabilization of p53 by NaBu allows it to binds to pro-apoptotic promoters and enhances their transcription (J. Chen et al., 2019).
It is worth mentioning that, the combination treatment showed a highly significant elevation in p53 gene more than each drug alone. It could be suggested that, despite the ability of abemaciclib to reduce AKT phosphorylation, and NF-κB protein level which are correlated to inhibition of MDM2 activity and expression respectively, it failed to stabilize p53 protein. While upon adding NaBu in combination with abemaciclib, p53 stabilization was augmented through inhibition of MDM2 recruitment facilitated by inhibition of HDAC1 activity. This combination would be highly beneficial in MDA-MB-231 cancer cells as these cells are reported to show overexpression of both MDM2 (Gao et al., 2019) and HDAC1 (Park et al., 2011). Stabilization of p53 was observed even with low doses combination, thus the lower doses combination could be recommended to maintain the cytocidal effect while preventing the large doses’ side effects.
The effect of each single drug or the combinations on p16 was investigated. p16 is an endogenous CDK4/6 inhibitor which regulates cell cycle progression in normal cells as it act as cell cycle brake (Vermeulen et al., 2003). Cancer cells rely on attenuating those cell cycle brakes to ease the cell proliferation process during tumorogenesis (Vermeulen et al., 2003). It was reported that MDA-MB-231 shows a low expression level of p16 (de Oliveira et al., 2016). On the other hand, overexpression of p16 could be implicated in tumorogenesis, by inducing genomic instability which might lead to additional mutation as KRAS mutation. Overexpression of p16 may also lead to a decrease in cell dependence on CDK4/6 and overexpression of cyclin E and CDK2 to complete the cell cycle progression (Milde-Langosch et al., 2001). In addition, overexpressed p16 may translocate to the cytoplasm and enhance oncogenic biological functions (Milde-Langosch et al., 2001; Romagosa et al., 2011). Therefore, p16 overexpression is considered as one of the cell cycle specific mechanisms of resistance against CDK4/6 inhibitors (Pandey et al., 2019).
In the present study abemaciclib caused a significant elevation in p16 gene and protein expression level, On the other hand NaBu as a single drug had no effect on p16 expression level. Similarly, Pellizzaro,et al (2001) reported that NaBu caused no change in p16 expression level in NCI-H23 non-small cell lung cancer cell line (Pellizzaro et al., 2001) However, in colon cancer cells, butyrate caused an elevation in p16 expression. (Schwartz et al., 1998).
Combination treatment of NaBu and abemaciclib showed a significant increase in p16 gene and protein level in comparison with abemaciclib. This effect was evident only with the high dose combination. Thus using the low dose combination could be preferable as the high dose may enhance abemaciclib resistance through increase p16 level.
One of the characteristic features of TNBC, is its high ability to migrate to distant organs especially the brain and the lung and this contributes in making TNBC a lethal and aggressive breast cancer subtype. The molecular mechanism that underlie cell migration in TNBC is the low cadherin 3/4, and the high level of EMT biomarkers such as snail.. In the present study after 24h of treatment, the cell migration was significantly reduced in all treated groups with no differences between the single drugs and the combination treatment. (Ocana andPandiella, 2017). After 48h, NaBu and the combinations treatment showed a further decrease in cell migration in comparison with abemaciclib. The anti-migration effect was more pronounced in low dose combination supporting the beneficial value of low dose combination over high dose combination. Anti-metastatic effect of abemaciclib and NaBu was previously noted in Caco-2 colorectal cancer cells (Lee et al., 2020) and MDA-MB-231 cells respectively (Park et al., 2011)
The anti-migration effect of both drugs may be linked to their ability to reduce NF-κB and elevation of active RB protein level. NF-κB acts as a direct transcription activator of metalloproteinase 9 (MMP-9) (Wang et al., 2018). MMP-9 has the ability to degrade the extracellular matrix facilitating the migration of cells to the blood stream and/or lymph nodes (Wang et al., 2018). NF-κB also stabilizes snail protein (Xia et al., 2014) which acts as a direct transcription repressor of cell adhesion molecule E-cadherin. Also, Rb protein act as a direct transcription activator of E-cadherin (Engel et al., 2015). Butyrate exerts an additional mechanism, as it attenuates AP-1 complex formation via inhibition of MAPK/ERK1-2 (W. Huang et al., 2019). AP-1 complex is implicated in metastasis as it is a transcription factor that induces the expression of MMP-9. (Wang et al., 2018)
In conclusion, as TNBC is the most aggressive breast cancer subtype so the need to develop a novel therapeutic protocol is of a great importance. Combining NaBu with abemaciclib could be a new therapeutic avenue as it enhanced the anti-tumor activity of abemaciclib through epigenetic regulation of cyclin D1, Rb, and p53 expression level. The combination also showed augmented anti-metastatic effect which was more pronounced with low dose treatment. This combination could improve treatment outcome and patient survival, as well as achieving the most challenging issue in cancer treatment that is reducing the dose without losing the drug effectiveness.