Biguanides are a family of antidiabetic drugs with documented anticancer properties in preclinical and clinical settings. Despite intensive investigation, how they exert their therapeutic effects is still debated. Many studies support the hypothesis that biguanides inhibit mitochondrial complex I, inducing energy stress and activating compensatory responses mediated by energy sensors. However, a major concern related to this “complex” model is that the therapeutic concentrations of biguanides found in the blood and tissues are much lower than the doses required to inhibit complex I, suggesting the involvement of additional mechanisms. This comprehensive review illustrates the current knowledge of pharmacokinetics, receptors, sensors, intracellular alterations, and the mechanism of action of biguanides in diabetes and cancer. The conditions of usage and variables affecting the response to these drugs, the effect on the immune system and microbiota, as well as the results from the most relevant clinical trials in cancer are also discussed.

A key mechanism driving colorectal cancer (CRC) development is the upregulation of MYC and its targets, including ornithine decarboxylase (ODC), a master regulator of polyamine metabolism. Elevated polyamines promote tumorigenesis in part by activating DHPS-mediated hypusination of the translation factor eIF5A, thereby inducing MYC biosynthesis. Thus, MYC, ODC and eIF5A orchestrate a positive feedback loop that represents an attractive therapeutic target for CRC therapy. Here we show that combined inhibition of ODC and eIF5A induces a synergistic antitumor response in CRC cells, leading to MYC suppression. We found that genes of the polyamine biosynthesis and hypusination pathways are significantly upregulated in colorectal cancer patients and that inhibition of ODC or DHPS alone limits CRC cell proliferation through a cytostatic mechanism, while combined ODC and DHPS/eIF5A blockade induces a synergistic inhibition, accompanied to apoptotic cell death in vitro and in mouse models of CRC and FAP. Mechanistically, we found that this dual treatment causes complete inhibition of MYC biosynthesis in a bimodal fashion, by preventing translational elongation and initiation. Together, these data illustrate a novel strategy for CRC treatment, based on the combined suppression of ODC and eIF5A, which holds promise for the treatment of CRC.

The Hedgehog receptor, Patched1 (PTCH1), is a well-known tumour suppressor. While the tumour suppressor’s activity is mostly ascribed to its function as a repressor of the canonical Smoothened/Gli pathway, its C-terminal domain (CTD) was reported to have additional non-canonical functions. One of them is the reduction of autophagic flux through direct interaction with the Unc-51, like the autophagy activating kinase (ULK) complex subunit autophagy-related protein-101 (ATG101). With the aim of investigating whether this function of PTCH1 is important in cancer cell fitness, we first identified frameshift mutations in the CTD of PTCH1 in cancer databases. We demonstrated that those mutations disrupt PTCH1 interaction with ATG101 and increase autophagic flux. Using deletion mutants of the PTCH1 CTD in co-immunoprecipitation studies, we established that the 1309–1447 region is necessary and sufficient for interaction with ATG101. We next showed that the three most common PTCH1 CTD mutations in endometrial, stomach and colon adenocarcinomas that cause frameshifts at S1203, R1308 and Y1316 lack the ability to interact with ATG101 and limit autophagic flux, determined by bafilomycin A1-sensitive accumulation of the autophagy markers LC3BII and p62. We next engineered PTCH1 indel mutations at S1223 by CRISPR/Cas9 in SW620 colon cancer cells. Comparison of two independent clones harbouring PTCH1 S1223fs mutations to their isogenic parental cell lines expressing wild-type PTCH1 showed a significant increase in basal and rapamycin-stimulated autophagic flux, as predicted by loss of ATG101 interaction. Furthermore, the PTCH1 CTD mutant cells displayed increased proliferation in the presence of rapamycin and reduced sensitivity to glycolysis inhibitors. Our findings suggest that loss of the PTCH1-ATG101 interaction by mutations in the CTD of PTCH1 in cancer might confer a selective advantage by stimulating autophagy and facilitating adaptation to nutrient deprivation conditions.

Spermidine (SPD) and related polyamines are small polycationic molecules typically elevated in cancer cells, where their depletion suppresses tumor growth both in vitro and in vivo. Paradoxically, SPD has also been proposed as a dietary supplement for its potential health benefits, including cancer prevention, prompting considerable interest in elucidating its mechanisms of action. In vitro studies using cultured cancer cell lines treated with exogenous SPD have yielded conflicting results, with reports of enhanced proliferation, cytotoxicity, or modulation of autophagy. To address these discrepancies, we used polyamine-depleted colorectal cancer (CRC) cells to systematically evaluate SPD's effects across a range of concentrations. Following depletion with difluoromethylornithine, SPD exhibited a biphasic response: at low concentrations (100 µM) induced DHPS-independent cytotoxicity mediated by bovine serum amine oxidase (BSAO) activity in fetal bovine serum. High SPD doses transiently inhibited the autophagic flux, while low doses did not display any effect at all time points tested. The DHPS inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) suppressed SPD-induced proliferation at low concentrations and unexpectedly prevented cytotoxicity at high concentrations. Kinetic assays revealed that GC7 also inhibits BSAO in a non-competitive manner (Ki ≈ 300 nM), independent of DHPS. In silico docking analysis indicated that GC7 binds BSAO via non-covalent interactions, outside the topaquinone organic cofactor site. These findings clarify the concentration-dependent effects of SPD in CRC cells, reconcile conflicting in vitro data, and identify BSAO as a previously unrecognized target of GC7, providing new mechanistic insights into polyamine-driven cancer biology.