Molecular mechanism of MLCK1 inducing 5-Fu resistance in colorectal cancer cells through activation of TNFR2/NF-κB pathway

Colorectal cancer (CRC) is a common malignancy of the digestive system and is one of the most common cancers in the world. According to GLOBOCAN data released by the National Cancer Institute, more than 1.88 million new colorectal cancer cases and 915,880 CRC-related deaths occurred in 2020, accounting for approximately one-tenth of cancer cases and deaths, the third highest incidence of all cancers (10.0%) and second in mortality (9.4%) of all cancers[1]. 5-Fu is a classical chemotherapeutic agent for colorectal cancer, which plays a key role in regulating of cell cycle and promoting apoptosis [2]. However, many patients have become unresponsive to 5-Fu during treatment, leading to recurrence and metastasis, therefore, chemotherapy resistance to 5-Fu has become an urgent issue.

On the one hand, chemotherapy resistance in colorectal cancer is related to epigenetic mutations, and intestinal structures such as mucosal barrier and microflora on the other hand. Particular interest is directed to tumor microenvironment function, which are the more promising processes that will be the subject of much research in the future [3]. Maintenance of intestinal mucosal homeostasis requires intestinal barrier function. Barrier dysfunction is thought to promote the development of intestinal and systemic diseases [4]. Expression of myosin light chain kinase 1(MLCK1) is closely associated with deficiency of intestinal mucosal barrier function [5, 6]. MLCK1 is encoded by the MYLK gene, which is a 210-kDa protein originally identified in cultured cells, embryonic tissues and endothelium [7‐10], and the recruitment of MLCK1 to the perijunctional actomyosin ring (PAMR) affects the binding of tight junction-related proteins such as ZO-1, claudins, and then increases the permeability of epithelial tight junctions, thereby causing a reduction in intestinal barrier function (Fig. 1a). The recently discovered small molecule named divertin prevents the recruitment of MLCK1 to the PAMR, then reducing myosin light chain (MLC) phosphorylation and preventing the loss of intestinal barrier function [11]. MLCK1 expression plays an important role in the formation and progression of intestinal diseases by destroying the permeability of epithelial barrier [11, 12]. And it is associated with the production of pro-inflammatory cytokine in the inflamed intestinal tissues, such as TNF, INF and et al. [13, 14](Fig. 1a). Vitro models have been taken to show MLCK-mediated phosphorylation of myosin II regulatory light chain (MLC) was required for paracellular permeability increases that follow activation of Na + –nutrient cotransport [15], enteropathogenic E. coli infection [16], or TNF stimulation [17]. In addition, several recent reports have implicated the role of MLCK in animal models of inflammatory bowel disease (IBD) [12, 18, 19]. Moreover, the association between MLCK and colitis-associated cancer (CAC) has been reported that the specific role of NF-κB activation in the inflamed epithelia via TNFR2 signaling [12]. Finally, expression of MLCK increased para-cellular permeability in vitro [20, 21] and in vivo [22]. Therefore, MLCK is a key signaling node in physiological and pathophysiological regulation of epithelial tight junctions. Studies have shown that MLCK is a potential target for affecting intestinal barrier function, as well as for tumor therapy [11, 22]. However, the relative contributions of MLCK expression and chemotherapy resistance in colorectal cancers have not been defined and there’s very little report on this.

The involvement of the NF-κB pathway in tumor formation mechanisms, including tumor chemotherapy resistance and disease progression, has been widely reported in many researches [23]. An important mediator causing intestinal epithelial NF-κB activation is TNF-α, which is significantly elevated in the inflamed intestinal environment [24]. Specific upregulation of TNFR2 is observed in inflammatory intestinal epithelial cells and activates MLCK1-dependent dysregulation of tight junctions, leading to apoptosis-mediated barrier loss and experimental colitis [12] (Fig. 1a). The relationship between MLCK1 expression and TNFR2/NF-κB pathway is poorly understood.

Here we show the impaction and mechanism of MLCK1 in tumor therapeutic susceptibility, especially for chemotherapy resistance of colorectal tumor cells to 5-Fu. Analysis of data from TCGA showed a significant difference in MLCK1 expression between normal and colorectal cancer patients (p < 0.01) (Fig. 1b), but no significant correlation with overall survival (OS) (Fig. 1c) and disease-free survival (DFS) (Fig. 1d). Other than that, our experimental data may, in part, explain molecular mechanism of MLCK1 expression and TNFR2/NF-κB pathway in 5-Fu resistance of colorectal cancer cells, and these results need to be confirmed by additional experimental clinical studies.

Epithelial barrier loss is a critical component of acute and chronic gastrointestinal diseases, such as food allergy, celiac disease, infectious enterocolitis, IBD and even neoplasms [39]. At advanced stages, colitis progresses might result in malignant colorectal disease including colitis-associated carcinoma (CAC), through proven pathways such as autophagy and mucosal barrier disruption [40]. Importantly, intestinal inflammation caused by disruption of the intestinal mucosal barrier and bacterial invasion, which bring about inflammatory cytokine like NF-κB, TNF-α, and IL-6, inflammatory cell infiltration, all of these contribute to survival advantages to abnormal proliferation in cancer cells [41]. However, signaling pathways such as TNF, NF-κB and so on are stimulated by exposure of the tumor to radiation or chemotherapy drugs in the tumor microenvironment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. These may cause chemotherapy resistance [42, 43]. Our data suggest that preservation of the epithelial barrier may be beneficial in preventing carcinoma development and chemotherapy resistance.

Consistently, We show here that MLCK1 high expression can enhance resistance to 5-Fu in colorectal cancer cells and the sensitivity to 5-Fu was increased after knocking down the expression of MLCK1, that might be closely correlated to TNFR2/NF-κB pathway. Briefly, MLCK1 knockout might prevent chemotherapy resistance by recovering epithelial barrier loss and altering the tumor microenvironment(TME) through TNFR2/NF-κB pathway. However, we need more experimental studies to confirm this hypothesis. So far, researches have shown that long MLCK knockout in vivo, which has no apparent toxicity, is beneficial in immune-mediated colitis [12]. Others reported that enzymatic MLCK inhibition could not be used therapeutically because MLCK knockout leads to hypotension, bladder dysfunction, severe intestinal dysmotility, and death [44]. this This limitation overcame by development of inducible knockout mice with loxP sites flanking the MYLK sequence encoding the catalytic domain [45]. Thus, while long MLCK inhibition maybe useful in oncology treatment, loss of MLCK enzymatic activity in smooth muscle [45] and non-muscle [12, 46] cells would have unacceptable toxicities.

MLCK is a key signaling node in physiological and pathophysiological of epithelial tight junctions by regulating Tight Junction (TJ) protein Interactions and structure. Previous research reported that epithelial NF-κB activation plays a role in opening paracellular spaces through dysfunction of tight junction proteins such as occludin, claudin-1 and zona occludens-1 [47]. An increase in MLCK protein expression in vitro and in vivo mediated TNF-induced barrier loss [31, 48]. However, in vitro studies from two groups conflict as to whether TNF-induced MLCK upregulation depended on NF-κB or AP-1 signaling [29, 30, 37]. A group found that the NF-κB inhibitors such as curcumin prevented TNF-induced MLCK upregulation and barrier loss [38, 49]. But another group found that a series of NF-κB inhibitors, such as curcumin, MG132, capsaicin and triptolide were unable to prevent TNF-induced barrier loss [14]. Interestingly, this group found that sulfasalazine was able to block TNF-induced barrier loss in a dose dependent manner which prevent barrier loss at low dose but exaggerate barrier loss at high dose [14].

The human long MLCK promoter has been cloned by different research groups, which contained functional binding sites for both NF-κB and AP-1 [29, 37]. In the common model, undifferentiated intestinal epithelial cells modestly upregulate long MLCK via NF-κB signaling, while differentiated intestinal epithelial cells upregulate long MLCK induced by TNF via AP-1 signaling. Additionally, one group has shown that AP-1-activating elements such as MAP kinases, are involved in TNF-induced long MLCK upregulation [50, 51]. These data might separate NF-κB from MLCK upregulation and suggest that another signaling pathway activated by TNF was responsible for barrier loss. At this point, the relationship between MLCK, NF-κB and TNF is becoming more clear which also provides a basis for the reversal of MLCK-related barrier loss by pharmacotherapy such as TNF inhibitor, NF-κB inhibitor, specific MLCK1 inhibitor and other inhibitors of inflammatory factors.

More interestingly, recent observation reported that an enzymatically inactive region, IgCAM3 [52], was responsible for long MLCK1 recruitment to the PAMR, blocking MLCK-dependent tight junction regulation in intestinal disease. They found a molecule, Divertin, was able to displace long MLCK1 from the PAMR, reverse TNF-induced barrier loss in vivo and in vitro [11]. Thus, divertin might be useful to reverse and prevent acute TNF-induced MLC phosphorylation and barrier loss in inflammatory diseases and play important role in treatment of malignant of the intestine. Conclusively, despite the difficulties in the treatment of intestinal malignancies have been overcome in the past few years, there remains much to be learned. Specific topics such as elucidation of mechanisms that regulate MLCK1 in loss of epithelial barrier function and further definition of pore pathway function in health and disease, and characterization of the structural and functional properties of defined and to-be-discovered in molecular mechanism of MLCK1 in preventing colorectal cancer chemotherapy resistance, which need more time and practice to confirm.