KRAS signalling and its cross-talk with other pathways have been reported associated with several cancers. In most PCa cases, the KRAS pathway and associated downstream pathways are found to have role in PCa development, progression, and poor prognosis. KLF13 has been demonstrated to have a role in PCa development. Further, it acts as a negative modulator for KRAS. KLF family members interact and bind with sin3A (co-transcription factor), facilitating their binding with DNA or histone modulators required to repress or activate gene expression. However, the interaction between KLF13 and sin3A is not studied. Therefore, in the present study, KLF13’s molecular interaction with co-repressor, Sin3A was evaluated, and the relative expression of KLF13 and its target genes was also studied.
To understand the molecular interaction between KLF13 and sin3A, molecular docking of both proteins must be performed. The protein data bank (PDB) search revealed that the KLF13 structure is not previously determined; therefore, the design of KLF13 was predicted through both the ab initio and threading approach. Obtained structures were evaluated through the ERRAT score, Ramachandran plot analysis, and superimposition approach and the most accurately predicted structure was selected. Furthermore, multiple sequence alignment was performed to find the unique features of KLF13 that also revealed the sin3A binding domain at its N-terminus. It interacts with sin3A through the AAECL sequence [46]. KLF11 recruits sin3A to the promoter region of SMAD7 and halts TGFβ signalling. In most cancers, the sin3A binding domain of KLFs is mutated which leads to cell proliferation [55].
KLF13 functions as a transcription repressor and has been reported to inhibit the expression of HMGCS1, Akt, and cAMP pathway-associated genes such as PRKACA, RAP1A ADCY6, and CALM3 and modulates cholesterol biosynthesis, cell growth and survival, and endocrine signalling [12, 56, 57]. KLFs interact and bind with the co-repressor Sin3A through its Sin3A-binding domain at N-terminus that facilitate KLF-Sin3A complex binding with HDAC in the nucleus [46, 51]. KLF13 possesses nuclear localization signal 147 LRQRVRRGRSRADLESPQRKHK 168 that allows its nuclear translocation [46]. It interacts with DNA through three zinc-finger DNA binding domains that particularly bind with the GC box or CTCCC box at the promoter region [58] [59]. KLF13 interaction with Sin3A is important for its binding with HDAC and inducing transcription repression. Studies have delineated the essentiality of Sin3A interaction with KLF13 for mediating its transcription regulator role in the nucleus [51]. However, no study has ever been performed to determine the molecular interaction between Sin3A and KLF13. Through a rigid docking approach, KLF13 and sin3A were docked and docked complex was subjected to molecular dynamics simulation. The outcome revealed that both hydrogen bonding and hydrophobic interactions participate in KLF13 and sin3A interaction. However, towards the end of the simulation hydrophobic interactions mainly contributed to the binding. This analysis provided an essential insight behind sin3A and KLF13 binding that can be exploited for designing KLF13 specific therapeutic agents.
Target genes of KLF13 that are also involved in KRAS signalling were identified through the HTFDB3.0 database [42]. The investigation revealed that KLF13 directly modulates the expression of oncogene TPD52 and PKCɛ and its family member KLF12 modulates the expression of miR-223. Therefore, in the present investigation, the differential expression of KLF13 with PKCɛ, miR-123, and TPD52 was investigated. KLF13 expression was found to be down-regulated in the blood of PCa patients. Like KLF13, the expression of miR-223 and PKCε was also down-regulated but TPD52 expression was up-regulated. The expression of TPD52 increased with advanced stages of cancer while miR-223 expression decreased with advanced stages of cancer. But the trend for KLF13 and PKCε expression was different. In comparison with a non-metastatic group, their expression was up-regulated in the metastatic group. Furthermore, the expression of PKCε was down-regulated in stage III/IV patients while KLF13 expression was down-regulated in the different stages of PCa in comparison to control but did not have any significant expression difference among them.
Previous research was performed to determine the independent expression and mechanism of action of these genes in cancers. Under current findings, elevated TPD52 expression and attenuated miR-223, KLF13 and PKCε expression is also reported by several studies [60]. Increased copy number of the TPD52 gene on the long arm of chromosome 8 is responsible for its elevated mRNA and protein levels in PCa [61]. In normal prostate tissue, its expression is regulated by the action of several tumour suppressor miRNAs such as miR-218, miR-224 and miR-103a. However, the down-regulation of translational modulators of TPD52 also promotes its aberrant expression [60, 62, 63]. Mechanistically, it activates Akt activation along with the activation of STAT3 and NF-κB by phosphorylating and causing RelA accumulation in the nucleus [64]. However, its expression attenuation is demonstrated in renal cell carcinoma (RCC). Ectopic TPD52 expression in RCC cell lines halted phosphorylation of PI3K and Akt, resulting in tumour growth inhibition and suppression of metastasis [53]. This suggests the tissue-specific role of TPD52.
The tumour suppressor role of KLF13 is demonstrated in the current study. A Similar was reported by Wang et al., who said KLF13 induced negative regulation of Akt [12]. Along with PCa, its downregulated expression is also written in gliomas and colorectal cancer [13, 65]. But KLF13 elevated expression is found in oral cancer [66]. In gliomas, KLF13 attenuated expression is brought about by DNMT1 action. It hypermethylated the KLF13 promoter region and inhibits its transcription [13].
In PCa, PKCε regulates the activation of Akt and ERK1/2 by promoting protein kinase D (PKD) nuclear translocation [67], suggesting an oncogenic role of PKCε. Similarly, PKCε oncogenic role is also demonstrated by Gutierrez-Uzquiza and team [68]. They reported that it contributes to mediating bone metastasis in PCa by inducing IL1β activity. In the present study, PKCε expression was decreased in the non-metastatic group, but its expression elevation was observed in the metastatic group. This expression trend of PKCε somehow hints toward its participation in metastasis. However, further in vitro analysis is required to confirm further its role and mechanism of action in PCa metastasis.
Expression of miR-223 is reported to decrease in gastric, glioblastoma, and bladder cancer [69–71]. The outcomes of these studies and the current study confirm the tumour suppressor role of miR-223 in cancers. Expression studies and in silico analysis have highlighted integrin A3 (ITGA3) and integrin B1 (ITGB1) as potential targets in PCa [72]. However, studies also reported ERG as a target of miR-223 in PCa [73]. MiR-223, by targeting these genes’ transcripts, promote PCa cell proliferation inhibition and apoptosis [72]. MiR-223, along with PAX6, regulates PI3K/Akt signalling and modulates chemoresistance and cancer stemness in glioblastoma [74].
The role of KLF13, TPD52, PKCɛ, and miRNA-223 in modulating signal transduction through Akt signalling was also highlighted through pathway analysis. Upstream and downstream targets of each gene are found by STRING software which sheds light on the unique crosstalk of Akt and Kras pathways through KLF13. Studies have identified KLF family members’ role as KRas suppressors [11]. Similarly, KLF13 contribution in inducing the inactivation of Akt is also reported [12]. Our study demonstrated the down-regulation of KLF13 in PCa, suggesting that KLF13 decreased expression promotes PCa cell progression by inducing signalling through Akt and KRas pathways. This further indicates that KLF13 can be a potential target for therapeutic approaches of PCa.