لخّصلي

Intense efforts are underway to develop synthetic Wnt signaling modulators, including small molecules (XAV939, pyrvinium), peptides (SFRP1), and blocking antibodies. FDA-approved lithium chloride stimulates CTNNB1, while NSAIDs like celecoxib inhibit CTNNB1-dependent transcription. Wnt-blocking antibodies show promise in targeting colon cancer and HCC cells, with WNT3A-neutralizing antibodies reducing prostate cancer proliferation in vivo. Regarding TGF-β signaling, while no definite clinical role exists, SMAD4 expression correlates with prognosis and 5-FU response. Clinical trials investigate 18qLOH for adjuvant therapy and the impact of chronic NSAID use on Notch pathway activity. Several clinical trials target pathways including IGF-1R, Wnt, Notch, Hedgehog, and TGF-β, with some showing promise (e.g., γ-secretase inhibitor RO4929097, vismodegib). MSI status is a reliable biomarker for immunotherapy response, with checkpoint inhibitors like pembrolizumab and nivolumab showing efficacy in metastatic CRC. Combination therapies (e.g., pembrolizumab/ipilimumab, nivolumab/ipilimumab) are also being investigated. Other PD-1/PD-L1 inhibitors are in phase I trials. Targeting the CIMP pathway involves anti-EGFR monoclonal antibodies (cetuximab, panitumumab) and TKIs. Cetuximab is effective but ineffective in tumors with RAS, BRAF, or PIK3CA mutations. Panitumumab shows improved OS and PFS in combination with FOLFOX or 5-FU/LV. There are no approved therapies for KRAS-mutated CRC, but AMG 510, targeting KRASG12C, shows promise, along with other agents targeting G12C and G12D mutations. While BRAF inhibitors have shown limited success alone in CRC, combinations with EGFR inhibitors (e.g., vemurafenib/cetuximab, encorafenib/cetuximab/alpelisib, dabrafenib/trametinib/panitumumab) show improved outcomes. Anti-VEGF/VEGFR therapies, such as bevacizumab, improve PFS and OS in metastatic CRC, including in KRAS-mutant patients. Further research is needed to clarify the role of molecular classification in therapeutic interventions and to identify reliable biomarkers for CRC risk. The future of CRC treatment likely involves personalized medicine using novel drug combinations and specific targets to minimize toxicity. Understanding CRC's underlying mechanisms will be crucial for developing novel diagnostic and therapeutic strategies.

There are intense efforts to develop synthetic modulators of Wnt signaling including small molecules, peptides, and blocking antibodies to inhibit Wnt pathway [175]. As approved by US Food and Drug Administration (FDA), lithium chloride is already in clinical use and it is found to stimulate CTNNB1 by inhibiting GSK3. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs) and celecoxib, the selective COX-2, block CTNNB1-dependent transcription in CRC [176,177] and lessen polyp formation in FAP patients as well in in vivo mice models of colon cancer [178,179]. Recently, two small molecules, XAV939 and pyrvinium, were identified using reporter-based screening approaches, while XAV939 increased AXIN stability by inhibiting tankyrase, pyrvinium stimulated CTNNB1 phosphorylation through casein kinase activation [180,181]. On the other hand, several Wnt-blocking antibodies blocked proliferation and induce apoptosis in different cancers [182]. In comparison to normal tissues, FZD7-specific antibodies selectively target colon cancer and HCC cells [183,184]. In vivo studies have shown that intraperitoneal injections of WNT3A-neutralizing antibodies reduce proliferation and increase apoptosis of prostate cancer in mouse models [185]. In addition, studies have indicated that use of Wnt-modulatory peptides, SFRP1, or SFRP1-derived peptides reduce HCT116 xenograft tumor development in nude mice [186,187].
On the other hand, no definite clinical role for TGF-β signaling pathway has been identified thus far; however, studies show that SMAD4 expression levels correlate with prognosis and response to 5-fluorouracil (5-FU) [188,189,190]. Moreover, current clinical trials (e.g., NCT00217737, also designated ECOG 5202) are studying the benefit of 18qLOH for directing the use of specific adjuvant therapies [72]. They also found that chronic use of NSAIDs, mainly Ibuprofen, stimulates a dose-dependent reduction of Notch pathway activity, thus confirming the protective effects of NSAIDs in CRC [72]. Remarkably, a few clinical trials aimed at targets including IGF-1R, Wnt, Notch, Hedgehog, and TGF-β are in progress; however, no definite results have appeared thus far. Phase II trials of the γ-secretase inhibitor (RO4929097) in Notch blockade therapy and the Hedgehog pathway inhibitor vismodegib can be considered promising [191,192].
3.2. Targeting MSI Pathway
MSI status has been demonstrated to be a reliable biomarker of immunotherapy response in the metastatic setting. Currently, checkpoint inhibitors are investigated in various solid tumors with promising responses [193,194]. Pembrolizumab, a humanized IgG4 antibody, was the first PD-1 blocker approved by FDA for treatment of metastatic CRC [193]. Another phase I trial in patients with MSI-H CRC found antitumor activity of pembrolizumab [194]. Although combination therapy of pembrolizumab and ipilimumab showed comparable efficacy in melanoma patients, the effect of this combination in CRC is still nascent [195]. The other humanized monoclonal IgG4-based PD-1 antibody, nivolumab, gained FDA approval for MSI-H metastatic CRC based on the results obtained in the CheckMate-142 clinical trial [196]. Remarkably, a combined therapy of nivolumab and ipilimumab improved the outcome of the patients with dMMR or MSI-H CRC who had previously received chemotherapy [197]. Other plausible candidate PD-1/PD-L1 inhibitors (atezolizumab, avelumab, and durvalumab) are under phase I trials for several tumors including CRC [198]. Furthermore, new immune checkpoint targets including TIM-3, TIGIT, T cell Ig, and T cell-derived LAG-3, which promote CRC progression, are also in phase I trials [199,200].
3.3. Targeting CIMP Pathway
Targeting the EGFR pathway generally involves use of anti-EGFR monoclonal antibodies and tyrosine kinase inhibitors (TKIs) aimed at intracellular kinases. Cetuximab was the first monoclonal antibody introduced to target EGFR. Cetuximab provokes EGFR internalization and degradation once bound to the external domain of EGFR [201]. Multiple studies have confirmed the beneficial effects of the cetuximab on CRC patients’ outcome [202]; in addition, a combined therapy of cetuximab with other existing chemotherapies also showed favorable results [203]. However, tumors carrying RAS, BRAF, or PIK3CA mutations, loss of PTEN, HER-2 amplification, and altered VEGF and VEGFR signaling are resistant to anti-EGFR therapy due to continuous activation of EGFR downstream signaling pathways [204]. In these cases, the patient is not eligible for anti-EGFR treatment with cetuximab. The other EGFR target, panitumumab, is a fully humanized antibody and, in comparison to cetuximab, does not provoke antibody-dependent cell-mediated cytotoxicity [205]. In the PRIME trial, efficacy of FOLFOX (folinic acid, fluorouracil, and oxaliplatin) alone as well as in combination with panitumumab was analyzed in patients with metastatic CRC. The combined therapy showed a higher OS and PFS than for FOLFOX alone [206,207]. PRIME and PEAK trials further analyzed the effects of panitumumab and fluorouracil/leucovorin (5-FU/LV) after panitumumab plus FOLFOX. The trials showed significant improvement in PFS and OS compared with panitumumab treatment alone [208]. In addition, the VALENTINO trial demonstrated synergistic effects of panitumumab with 5-FU/LV and better effects on outcome compared with treatment with panitumumab alone [209]. Although both cetuximab and panitumumab are FDA-approved drugs, panitumumab is economically more effective than cetuximab [210].
On the other hand, there are no approved specific targeted therapies for KRAS-mutated CRC. However, a novel agent, AMG 510, which is a small molecule that targets KRASG12 C mutation, was introduced. AMG 510 specifically and irreversibly blocks KRASG12 C by locking it in the inactive GDP-bound state [211]. In addition, other KRAS-modulating agents targeting G12C (MRTX849, LY3499446, or ARS-1620) and G12D mutations are also under investigations [212,213].
Although BRAF mutations are more frequent in melanoma and papillary thyroid carcinoma in comparison with CRC, a few studies analyzed the effects of BRAF inhibitors (vemurafenib or dabrafenib and trametinib) in patients with metastatic CRC. Although downstream MAPK activity was inhibited, the PFS or OS of patients did not improve [214,215,216]. However, the synergistic effect of BRAF and EGFR inhibitors in trials using vemurafenib in combination with IRI and cetuximab in BRAF-mutant CRC patients showed positive results [217,218,219,220]. Additionally, in a phase II trial, using encorafenib (a BRAF inhibitor) along with cetuximab, with or without alpelisib (ALP), improved the OS and PFS in advanced BRAF-mutant CRC patients [221]. Furthermore, a study by Corcoran et al. [218] found that patients with BRAF-V600E-mutated CRC, when treated with a triplet regimen (dabrafenib, trametinib, and panitumumab), produced a better response rate in comparison with the double regimens (dabrafenib + panitumumab or trametinib + panitumumab). The ongoing BEACON trial, which employs the triplet regimen of encorafenib, binimetinib (a MEK inhibitor), and cetuximab, has shown less toxicity and higher survival rates [222].
Anti-VEGF/VEGFR therapies are necessary to target steps in tumor metastasis. Bevacizumab (Avistin), a humanized IgG monoclonal antibody, was the first FDA-approved VEGF-targeted agent for metastatic CRC. According to several trials, bevacizumab showed to improve both progression-free survival and OS in metastatic CRC [223,224]. Furthermore, KRAS-mutant patients were also found to benefit from bevacizumab [225,226]. Moreover, combination of FOLFOX and bevacizumab improved progression-free survival and OS in CRC patients as compared to treatment with FOLFOX alone [227].
4. Conclusions
This brief review provides information about candidate biomarkers that can aid in improving the diagnosis and help with the early detection of CRC cases, thus ameliorating the prognosis of CRC patients. Although there are marked advances in CRC investigations, the role of molecular classification in therapeutic interventions remains unclear. The use of molecular alterations in predicting risk for CRC shows promise, while further studies are needed to determine if aberrantly methylated CpGs or other molecular alterations can be used as reliable and accurate indicators of risk for polyps or CRCs. Moreover, it is important to analyze the efficacy of multi-kinase/BRAF-inhibitor, sorafenib, in addition to other specific inhibitors of the EGFR as well as PI3K signaling pathway, in the treatment of CRC to further identify novel therapeutic targets. On the other hand, we believe that using new drug combinations and specific (personalized) targets will be the future avenue for efficient treatment of CRC patients with limited risk of toxicity and adverse side effects. Thus, understanding the underlying mechanisms of CRC cell genetic alteration and subsequent consequences can help pave the way for the development of novel diagnostic and therapeutic strategies.