Biology subjects

Wisniewski, D. J.

Publications and source records attributed to Wisniewski, D. J..

3 recordsLinked to original sources

bioRxiv2026

Loss of Flotillin-2 enhances trastuzumab emtansine internalization and cytotoxicity by relieving negative regulation of HER2 internalization in HER2-amplified cancers

While Trastuzumab emtansine (T-DM1) and other HER2-targeting antibody-drug conjugates (ADCs) are used to treat cancer patients with HER2-amplified tumors, there is a need to improve the efficacy through the understanding of their mechanism of uptake into cells. Flotillin-2 (FLOT2) regulates the internalization of epidermal growth factor receptor (EGFR), leading us to investigate FLOT2 effects on HER2 internalization. Higher FLOT2 expression in nine HER2 amplified cell lines correlated with a higher T-DM1 IC50 in vitro, and breast cancer patients with high FLOT2 expression had worse survival when receiving either T-DXd (16.2 months (m) vs 18.3 m, p=0.04) or T-DM1 (38.0 m vs 41.3 m, p=0.1) in real-world Caris Life Sciences data. FLOT2 interacts with HER2 and positively regulates HER2 activation and downstream signaling, while FLOT2 knockdown reduces the viability of HER2 amplified cancer cells. FLOT2 knockdown results in increased HER2 internalization upon binding of T-DM1, mediated by ubiquitination by the Cbl ubiquitin ligases. We investigated the effects of various small molecules and discovered that zoledronic acid binds to FLOT2 and disrupts the HER2/FLOT2 interaction, which enhances T-DM1 internalization and cytotoxicity. In conclusion, FLOT2 regulates the internalization and cytotoxicity of T-DM1 mediated by Cbl-dependent ubiquitination of HER2. Zoledronic acid disrupts the HER2/FLOT2 interaction, therefore increasing the internalization and cytotoxicity of T-DM1, providing proof of principle that a small molecule inhibitor of the HER2/FLOT2 interaction can enhance the activity of the HER2-targeting ADC.

cancer biology↗

bioRxiv2025

Dual inhibition of EGFR and PI3Kinase signaling in EGFR amplified triple negative breast cancer cells induces apoptosis and reduces tumor growth

EGFR family receptor tyrosine kinase signaling is commonly dysregulated in cancer by amplification or activating mutations. Although studies have investigated dual EGFR/PI3K inhibition in breast cancer, they have not determined biomarkers which predict success. We present evidence of a patient subset with EGFR amplification and PI3Kinase pathway mutations in breast cancer which can be synergistically targeted by dual EGFR/PI3K inhibition. This study identified that EGFR amplification occurs in approximately 1-5% of breast cancer patients with shorter overall survival compared to unamplified patients. Up to 71% of EGFR amplified tumors have activating mutations in the PI3K pathway. Dual EGFR/PI3K inhibition more dramatically reduced mTOR and AKT signaling in BT20 and MDA-MB-468 cells which both have EGFR amplification and PI3K pathway activating mutations, compared to control cells. Dual inhibition synergistically reduced cell viability and increased apoptosis in MDA-MB-468 and BT20 compared to control. Single agent therapy in a BT20 xenograft model reduced tumor volume, however only the combination statistically significantly reduced tumor volume compared to control. We conclude that EGFR amplification with co-incident PI3K pathway mutations are driver mutations in a subset of breast cancers and present a subgroup of breast cancers that are more likely to respond to dual targeted therapy.

cancer biology↗

bioRxiv2022

Flotillin-2 regulates EGFR activation, degradation, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Overexpression of FLOT2 decreased EGFR sjgnaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

cancer biology↗