Targeting Mitochondria with ClpP Agonists as a Novel Therapeutic Opportunity in Breast Cancer.

Breast cancer is the most frequently diagnosed malignancy worldwide and the leading cause of cancer mortality in women. Despite the recent development of new therapeutics including targeted therapies and immunotherapy, triple-negative breast cancer remains an aggressive form of breast cancer, and thus improved treatments are needed. In recent decades, it has become increasingly clear that breast cancers harbor metabolic plasticity that is controlled by mitochondria. A myriad of studies provide evidence that mitochondria are essential to breast cancer progression. Mitochondria in breast cancers are widely reprogrammed to enhance energy production and biosynthesis of macromolecules required for tumor growth. In this review, we will discuss the current understanding of mitochondrial roles in breast cancers and elucidate why mitochondria are a rational therapeutic target. We will then outline the status of the use of mitochondria-targeting drugs in breast cancers, and highlight ClpP agonists as emerging mitochondria-targeting drugs with a unique mechanism of action. We also illustrate possible drug combination strategies and challenges in the future breast cancer clinic.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry 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 EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (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. Together, 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.

Fatty acid synthase mediates high glucose-induced EGFR activation in oral dysplastic keratinocytes.

BACKGROUND: Recent studies point to the epidermal growth factor receptor (EGFR) as a critical mediator of type 2 diabetes mellitus (T2DM)-induced renal, cardiac, and ocular complications. T2DM is considered a systemic contributing factor in oral carcinogenesis. Similarly, increased EGFR gene copy number and protein expression strongly predict tumor progression. Yet, the impact of hyperglycemia on EGFR activity in oral potentially malignant disorders remains unclear. We recently reported that fatty acid synthase (FASN), a key de novo lipogenic enzyme, mediates EGFR activation in nicotine-treated oral dysplastic keratinocytes. While in non-malignant tissues FASN expression is extremely low, it is frequently upregulated in several cancers, including oral squamous cell carcinoma. The present study was carried out to investigate whether high glucose conditions trigger pro-oncogenic responses in oral dysplastic keratinocytes via FASN-mediated EGFR activation. METHODS: Cell viability and migration of oral dysplastic keratinocytes were evaluated when exposed to normal (5 mM) or high (20 mM) glucose conditions in the presence of FASN and EGFR inhibitors. Western blotting was also performed to assess changes in FASN protein expression and EGFR activation. RESULTS: Oral dysplastic keratinocytes exposed to high glucose led to EGFR activation in a FASN-dependent manner. Likewise, high glucose significantly enhanced cell viability and migration in a FASN/EGFR-mediated fashion. Notably, EGFR inhibition by the anti-EGFR monoclonal antibody cetuximab significantly reduced the proliferation of FASN-overexpressing oral dysplastic keratinocytes. CONCLUSION: These novel findings suggest that FASN may act as a key targetable metabolic regulator of glucose-induced EGFR oncogenic signaling in oral potentially malignant disorders.

Nicotine induces oral dysplastic keratinocyte migration via fatty acid synthase-dependent epidermal growth factor receptor activation.

Despite advances in diagnostic and therapeutic management, oral squamous cell carcinoma (OSCC) patient survival rates have remained relatively unchanged. Thus, identifying early triggers of malignant progression is critical to prevent OSCC development. Traditionally, OSCC initiation is elicited by the frequent and direct exposure to multiple tobacco-derived carcinogens, and not by the nicotine contained in tobacco products. However, other nicotine-containing products, especially the increasingly popular electronic cigarettes (e-cigs), have unknown effects on the progression of undiagnosed tobacco-induced oral premalignant lesions, specifically in regard to the effects of nicotine. Overexpression of fatty acid synthase (FASN), a key hepatic de novo lipogenic enzyme, is linked to poor OSCC patient survival. Nicotine upregulates hepatic FASN, but whether this response occurs in oral dysplastic keratinocytes is unknown. We hypothesized that in oral dysplastic keratinocytes, nicotine triggers a migratory phenotype through FASN-dependent epidermal growth factor receptor (EGFR) activation, a common pro-oncogenic event supporting oral carcinogenesis. We report that in oral dysplastic cells, nicotine markedly upregulates FASN leading to FASN-dependent EGFR activation and increased cell migration. These results raise potential concerns about e-cig safety, especially when used by former tobacco smokers with occult oral premalignant lesions where nicotine could trigger oncogenic signals commonly associated with malignant progression.