Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography-mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

Breast cancer stem cells are involved in Trastuzumab resistance through the HER2 modulation in 3D culture.

Breast cancer human cells culture as spheroids develop autophagy and apoptosis, which promotes Trastuzumab resistance in HER2 overexpressing cells. Our aim was to study the association of the hostile environment developed in 3D with the breast cancer stem cells population and the HER2 modulation. Human mammary adenocarcinoma cell lines were cultured as spheroids using the hanging drop method. We generated hypoxia conditions by using a hypoxic chamber and CoCl2 treatment. Breast cancer stem cells were measured with mammosphere assays, the analysis of CD44 + CD24low population by flow cytometry and the pluripotent gene expression by RT-qPCR. HER2 expression was evaluated by flow cytometry and Western blot. MTS assays were conducted to study cell viability. Hostil environment developed in spheroids, defined by hypoxia and autophagy, modulated the response to Trastuzumab. In HER2+ cells with acquired resistance, we observed an increase in the breast cancer stem cell population. In BT474 spheroids, Trastuzumab induced the acquisition of resistance, along with an increase in breast cancer stem cells. Also, in 3D culture conditions we determined a modulation in the HER2 expression. Moreover, breast cancer stem cells showed enhanced HER2 expression. Finally, cells without HER2 gene amplification cultured as spheroids were sensitive to Trastuzumab, diminishing HER2 expression and cancer stem cells. Our findings show that 3D architecture is able to modulate breast cancer stem cell population and HER2 distribution, modifying the cell response to Trastuzumab.

Oral-specific ablation of Klf4 disrupts epithelial terminal differentiation and increases premalignant lesions and carcinomas upon chemical carcinogenesis.

BACKGROUND: Squamous cell carcinoma (SSC) of the head and neck is the sixth most common cancer and is rarely diagnosed in early stages. The transcription factor Krϋppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation. Inducible mice carrying an oral-specific ablation of Klf4 (K14-CreER(tam) /Klf4(flox/flox) ) develop mild dysplastic lesions and abnormal differentiation in the tongue. Aiming to analyze whether Klf4 cooperate in oral chemical carcinogenesis,we applied 4-nitroquinoline 1-oxide (4NQO), a tobacco surrogate, to this conditional Klf4 knockout mice. METHODS: K14-CreER(tam) /Klf4(flox/flox) and control mice were treated with 4NQO for 16 weeks and monitored until week 30. Histopathological samples were used for diagnostic purposes and immunofluorescence detection of epithelial differentiation markers. RESULTS: 4NQO-treated K14-CreER(tam) /Klf4(flox/flox) mice (Klf4KO 4NQO) showed a significant weight loss and developed more severe dysplastic lesions than control mice with 4NQO (P < 0.005). The Klf4KO 4NQO showed a tendency to higher incidence of oral SCC and a marked keratinization pattern in dysplasias, in situ carcinomas and SCC. Also, tongues derived from Klf4KO 4NQO mice exhibited reduced terminal differentiation as judged by cytokeratin 1 staining when compared with 4NQO-treated controls. CONCLUSIONS: Klf4 ablation results in more severe dysplastic lesions in oral mucosa, with a tendency to higher incidence of SCC, after chemical carcinogenesis. We show here, in a context similar to the human carcinogenesis, that absence of Klf4 accelerates carcinogenesis and correlates with the absence of cytokeratin 1 expression. These results suggest a potential role for KLF4 as a tumor suppressor gene for the tongue epithelium.

Impairing squamous differentiation by Klf4 deletion is sufficient to initiate tongue carcinoma development upon K-Ras activation in mice.

Oral squamous cell carcinoma (SCC) is among the most prevalent cancers in the world and is characterized by high morbidity and few therapeutic options. Like most cancers, oral SCC arises from a multistep process involving alterations of genes responsible for balancing proliferation and differentiation. Among these, Krϋppel-like factor 4 (Klf4) suppresses cell proliferation and promotes differentiation and thus helps to maintain epithelial homeostasis. However, the prevailing role of Klf4 in maintenance of normal homeostasis in oral epithelium has not been established in vivo. Here, we used an inducible oral-specific mice model to selectively ablate Klf4 in the oral cavity. We generated K14-CreER(Tam)/Klf4 (f/f) mice that survived to adulthood and did not present overt phenotype. However, histologically these mice showed dysplastic lesions, increased cell proliferation and abnormal differentiation in the tongue 4 months after induction, supporting a homeostatic role of Klf4 in the oral epithelia. Furthermore, by breeding these mutants with a transgenic line expressing at endogenous levels K-ras (G12D), we assessed the role of disrupting differentiation gene programs to the carcinogenesis process. The K14-CreER(TAM)/K-ras (G12D)/Klf4 (-) (/-) mice rapidly develop oral SCC in the tongue. Thus, our findings support the emerging notion that activation of differentiating gene programs may represent a barrier preventing carcinogenesis in epithelial cells harboring oncogenic mutations, and thus that molecules acting upstream and downstream of Klf4 may represent components of a novel tumor-suppressive pathway.