The Class III β-tubulin isotype (βIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while βIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.

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Class III β-tubulin plays a prominent role in the development of drug resistance to paclitaxel by allowing the incorporation of the GBP1 GTPase into microtubules. Once in the cytoskeleton, GBP1 binds to prosurvival kinases such as PIM1 and initiates a signaling pathway that induces resistance to paclitaxel. Therefore, the inhibition of the GBP1:PIM1 interaction could potentially revert resistance to paclitaxel. A panel of 44 4-azapodophyllotoxin derivatives was screened in the NCI-60 cell panel. The result is that 31 are active and the comparative analysis demonstrated specific activity in paclitaxel-resistant cells. Using surface plasmon resonance, we were able to prove that NSC756093 is a potent in vitro inhibitor of the GBP1:PIM1 interaction and that this property is maintained in vivo in ovarian cancer cells resistant to paclitaxel. Through bioinformatics, molecular modeling, and mutagenesis studies, we identified the putative NSC756093 binding site at the interface between the helical and the LG domain of GBP1. According to our results by binding to this site, the NSC756093 compound is able to stabilize a conformation of GBP1 not suitable for binding to PIM1.

Extracellular vesicles (EVs) have become a key tool in the biotechnological landscape due to their well-documented ability to mediate intercellular communication. This feature has been explored and is under constant investigation by researchers, who have demonstrated the important role of EVs in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. Unfortunately, there are still some limitations to overcome before clinical application, including the inability to confine the EVs to strategically defined sites of interest to avoid side effects. In this study, for the first time, EV application is supported by 3D bioprinting technology to develop a new strategy for applying the angiogenic cargo of human umbilical vein endothelial cell-derived EVs in regenerative medicine. EVs, derived from human endothelial cells and grown under different stressed conditions, were collected and used as bioadditives for the formulation of advanced bioinks. After in vivo subcutaneous implantation, we demonstrated that the bioprinted 3D structures, loaded with EVs, supported the formation of a new functional vasculature in situ, consisting of blood-perfused microvessels recapitulating the printed pattern. The results obtained in this study favour the development of new therapeutic approaches for critical clinical conditions, such as the need for prompt revascularization of ischaemic tissues, which represent the fundamental substrate for advanced regenerative medicine applications.

Atherosclerotic plaque instability and rupture in patients with asymptomatic carotid artery stenosis (ACAS) is a leading cause of major adverse cardiac events (MACE). This could be mainly evidenced in patients with pre-diabetes. Indeed, the altered glucose homeostasis and insulin resistance could cause over-inflammation of atherosclerotic plaque, favoring its conversion to unstable phenotype with rupture and MACE. Notably, metformin therapy reducing the metabolic distress and the inflammatory burden could reduce MACE in ACAS patients with pre-diabetes. In this setting, the microRNAs (miRs) could be used as molecular biomarkers of atherosclerosis progression, plaque rupture, and worse prognosis in normoglycemics (NG) versus pre-diabetics metformin users (PDMU) versus pre-diabetics non-metformin users (PDNMU). However, our study aimed to investigate a wide miRNA panel in peripheral blood exosomes from patients with ACAS divided in NG versus PDMU versus PDNMU, and to associate the circulating miRNA expression profiles with MACE at 2 years of follow-up after endarterectomy. The study included 234 patients with ACAS divided into NG (n = 125), PDNMU (n = 73), and PDMU (n = 36). The miRs’ expression profiles of circulating exosomes were determined at baseline and at 2 years of follow-up by Affymetrix microarrays from the patients’ plasma samples from any study cohort. Then we collected and analyzed MACE at 2 years of follow-up in NG versus PDMU versus PDNMU. Prediabetics versus NG had over-inflammation (p < 0.05) and over expressed miR-24 and miR-27 at baseline. At 2 years of follow-up, PDNMU versus NG, PDMU versus NG, and PDNMU versus PDMU over-expressed inflammatory markers and miR-24, miR-27, miR-100, miR-126, and miR-133 (p < 0.05). Finally, at the end of follow-up, we observed a significant difference about MACE comparing PDNMU versus NG (n = 27 (36.9%) versus n = 8 (6.4%); p < 0.05), PDNMU versus PDMU (n = 27 (36.9%) versus n = 6 (16.6%); p < 0.05); and PDMU versus NG (n = 6 (16.6%) versus n = 8 (6.4%); p < 0.05). Admission glucose values (HR (hazard ratio) 1.020, CI (confidence of interval) 95% (1.001–1.038), p = 0.029), atheromatous carotid plaque (HR 5.373, CI 95% (1.251–11.079), p = 0.024), and miR-24 (HR 3.842, CI 95% (1.768–19.222), p = 0.011) predicted MACE at 2 years of follow-up. Specific circulating miRs could be over-expressed in pre-diabetics and specifically in PDNMU versus PDMU after endarterectomy. MiR24, hyperglycemia, and atheromatous plaque could predict MACE at 2 years of follow-up.