Biology subjects

Freddi, S.

Publications and source records attributed to Freddi, S..

7 recordsLinked to original sources

bioRxiv2026

Decoding EGFR ligand bias through an endocytic organelle platform

How growth factor receptors decode ligand identity into distinct cellular responses remains a fundamental question in cell signaling. Here, we identify a receptor-proximal mechanism that links ligand-specific EGFR activation to distinct endocytic and biological outputs. We show that EGF, but not TGF, selectively engages a RAC1-PLC{gamma}2-IP3R signaling axis that supports EGFR non-clathrin endocytosis (NCE). PLC{gamma}2, but not PLC{gamma}1, localizes to RTN3-dependent PM-ER contact sites, where it generates localized Ca{superscript 2} signals required for completion of NCE, mitochondrial activation and cell motility. This specificity requires the RAC-binding interface of PLC{gamma}2 and is associated with RAC1-dependent formation of CTxB-positive PM regions, indicating that spatial organization contributes to signaling specificity. TGF fails to efficiently assemble the EGFR-associated organelle platform and instead favors clathrin-dependent EGFR uptake, prolonged proliferative signaling, greater organoid yield, and reduced migration compared with EGF. Together, our findings identify the RAC1-PLC{gamma}2 axis as the key determinant that decodes EGFR ligand bias by coupling receptor trafficking to the metabolic program that supports cell migration.

cell biology↗

bioRxiv2026

Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

Aberrant endocytosis has long been associated with epithelial plasticity and tumorigenesis, but direct in vivo evidence of its causal role in tumor progression and metastasis has been lacking. Here, we identify and molecularly characterize a previously unrecognized form of E-cadherin (ECAD) internalization in mammary epithelial cells. This process is mediated by the endocytic adaptor Epsin 3 (EPN3) through glycolipid-lectin (GL-Lect) driven endocytosis requiring galectin-3 and Eps15-family adaptors. Leveraging an EPN3 knock-in mouse model, we show that dysregulation of GL-Lect driven endocytosis disrupts mammary gland morphogenesis and activates epithelial-to-mesenchymal plasticity (EMP), synergizing with the ERBB2/Neu breast oncogene to drive metastasis. Pharmacologic inhibition of the GL-Lect mechanism suppresses morphogenetic and invasive phenotypes ex vivo, providing proof-of-concept for therapeutic targeting. These findings establish the GL-Lect mechanism as a driver of metastatic plasticity and uncover a tractable vulnerability in BC.

cancer biology↗

bioRxiv2025

Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

NUMB is an endocytic protein with tumor suppressor activity, largely mediated by its ability to inhibit p53 degradation. This function depends on the inclusion of a short alternatively spliced exon (Ex3) in NUMB, although the mechanistic link between endocytosis and p53 regulation remains unclear. Here, we show that the Ex3-encoded sequence directs NUMB to the plasma membrane, where it forms a complex with the endocytic adaptor SNX9. This complex recruits p53 in a SNX9-dependent manner and is internalized and trafficked to multivesicular bodies, culminating in exosomal secretion, in a process requiring both SNX9 and NUMB. Exosomal p53 is taken up by recipient cells and translocated to the nucleus, where it activates p53-dependent transcriptional and phenotypic programs. These findings suggest that exosome-mediated p53 transfer may contribute to the establishment of a tumor-suppressive microenvironment.

cancer biology↗

bioRxiv2025

A TranSNP in the DDIT4 mRNA can impact its translation efficiency and modulate p53-dependent responses in cancer cells

Relatively few studies have examined the link between SNPs and mRNA translation, despite the established importance of translational regulation in shaping cell phenotypes. We developed a pipeline analyzing the allelic imbalance in total and polysome-bound mRNAs from paired RNA-seq data of HCT116 cells and identified 40 candidate tranSNPs, i.e. SNPs associated with allele-specific translation. Among them, the SNP rs1053639 (T/A) on DNA damage-inducible transcript 4 (DDIT4) 3UTR was identified, with the reference T allele showing a higher polysome association. rs1053639 TT clones generated by genome editing exhibited significantly higher DDIT4 protein levels than AA ones. The difference in DDIT4 proteins was even greater when cells were treated with Thapsigargin or Nutlin, two perturbations that induce DDIT4 transcription. The RNA-binding protein RBMX influenced these allele-dependent differences in DDIT4 protein expression, as shown by RNA-EMSA, RIP, and smiFISH assays. RBMX depletion reduced DDIT4 protein in TT clones to the AA levels. Functionally, TT clones more effectively repressed mTORC1 under ER stress, while AA clones outcompeted TT clones in vitro or when injected in zebrafish embryos. RBMX depletion increased the fitness of TT cells in co-culture experiments. The rs1053639 AA genotype, under a recessive model, correlates with poor prognosis in TCGA cancer data. Key points- Translatome analysis in HCT116 cells revealed allele-specific mRNA translation for 40 SNPs - rs1053639 (T/A) in DDIT4 3UTR showed allelic differences in mRNA localization & protein expression - AA cells showed weaker mTOR inhibition & higher proliferation; AA individuals had poorer prognosis

molecular biology↗

bioRxiv2025

The CRL7FBXW8 Complex Controls the Mammary Stem Cell Compartment Through Regulation of NUMB Levels

NUMB is a tumor suppressor gene that functions by inhibiting the action of the NOTCH proto-oncogene and enhancing the levels and activity of the tumor suppressor protein p53. In breast cancer (BC), NUMB loss-of-function (LOF), mediated by various molecular mechanisms, is a frequent and causal event. Herein, we establish that loss of NUMB protein, resulting from protein hyper-degradation, is the prevalent mechanism of NUMB LOF in BC. Through a RNAi-based screening, we identified the CRL7FBXW8 complex as the E3 ligase complex responsible for NUMB hyper-degradation in BC. Genetic and pharmacological inhibition of CRL7FBXW8 rescued the transformation-related phenotypes induced by NUMB LOF in BC cell lines and in patient-derived xenografts. These effects were directly dependent on the restoration of NUMB protein levels. Thus, enhanced CRL7FBXW8 activity, through its interference with the tumor suppressor activity of NUMB, is a causal alteration in BC, suggesting it as a potential therapeutic target for precision medicine.

cancer biology↗

bioRxiv2024

Loss of the tumor suppressor NUMB drives aggressive bladder cancer through hyperactivation of a RhoA/ROCK/YAP signaling circuitry

Bladder cancer (BCa) is one of the most challenging and costly cancers to treat, yet little progress has been made on the development of predictive biomarkers and targeted therapies. Here, we uncover a critical function of Numb as a tumor suppressor in the bladder, identifying loss of Numb expression as a causal alteration in BCa that underlies biological aggressiveness and disease progression. Through retrospective cohort studies, we established that a Numb-deficient tumor status correlates with worse overall survival in post-cystectomy muscle-invasive bladder cancer (MIBC) patients and increased risk of MIBC progression in non-muscle-invasive bladder cancer (NMIBC) patients. The prognostic value of Numb loss can be attributed to its crucial role as a determinant of aggressive bladder tumorigenesis, as demonstrated in mouse and human models. Targeted Numb ablation in the basal layer of the urothelium was alone sufficient to trigger spontaneous bladder tumorigenesis and drive progression from preneoplastic to preinvasive and, ultimately, overtly invasive tumors. Additionally, Numb ablation sensitized the urothelium to other oncogenic insults, accelerating tumor onset and progression. Using 3D-Matrigel organoid cultures to recapitulate bladder tumorigenesis in vitro, we found that Numb loss heightens the proliferative and invasive potential of both mouse and human BCa cells. Integrative transcriptomic and functional analyses revealed that downregulation of the canonical Hippo pathway, resulting in enhanced YAP transcriptional activity, underlies the biological aggressiveness of Numb-deficient BCa. These molecular events are dependent on the activation of RhoA/ROCK signaling subsequent to Numb loss. Thus, a dysfunctional Numb-RhoA/ROCK-Hippo/YAP regulatory network is at play in aggressive Numb-deficient BCa and represents a therapeutic vulnerability. A 27-gene prognostic signature capable of identifying high-risk Numb-deficient patients could provide the basis of a clinical tool to stratify patients for innovative RhoA/ROCK/YAP targeted therapies. One Sentence SummaryNumb loss-directed hyperactivation of RhoA/ROCK/YAP underlies aggressive bladder cancer biology.

cancer biology↗

bioRxiv2024

A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

cell biology↗