Integrin ß3 Reprogramming Stemness in HER2-Positive Breast Cancer Cell Lines.

HER2-positive breast cancer, characterised by overexpressed HER2 levels, is associated with aggressive tumour behaviour and poor prognosis. Trastuzumab is a standard treatment; however, approximately 50% of patients develop resistance within one year. This study investigates the role of ITGß3 in promoting stemness and resistance in HER2-positive breast cancer cell lines (HCC1599 and SKBR3). The findings demonstrate that chronic exposure to trastuzumab upregulates stem cell markers (SOX2, OCT4, KLF4, NANOG, SALL4, ALDH, BMI1, Nestin, Musashi 1, TIM3, CXCR4). Given the documented role of RGD-binding integrins in drug resistance and stemness, we specifically investigated their impact on resistant cells. Overexpression of ITGß3 enhances the expression of these stem cell markers, while silencing ITGß3 reduces their expression, suggesting a major role for ITGß3 in maintaining stemness and resistance. Further analysis reveals that ITGß3 activates the Notch signalling pathway, known for regulating stem cell maintenance. The combination of trastuzumab and cilengitide, an integrin inhibitor, significantly decreases the expression of stem cell markers in resistant cells, indicating a potential therapeutic strategy to overcome resistance. These results identify the importance of ITGß3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy.

RIPK4 suppresses the TGF-ß1 signaling pathway in HaCaT cells.

Receptor-interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor-ß 1 (TGF-ß1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF-ß signaling during epidermal homeostasis-related events and suppression of RIPK4 expression by TGF-ß1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor-κB (NF-κB), protein kinase C (PKC), wingless-type MMTV integration site family (Wnt), and (mitogen-activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad-mediated TGF-ß1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad-mediated TGF-ß1 signaling events through suppression of TGF-ß1-induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3-Smad4 interaction, nuclear localization, and TGF-ß1-induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound-scratch assay demonstrated that RIPK4 suppressed TGF-ß1-mediated wound healing through blocking TGF-ß1-induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF-ß1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF-ß1 signaling.

Keratin 14 is a novel interaction partner of keratinocyte differentiation regulator: receptor-interacting protein kinase 4.

The epidermis, the outer layer of the skin, is formed by stratified keratinocyte layers. The self-renewal of the epidermis is provided by sustained proliferation and differentiation of the keratinocyte stem cells localized to the basal layer of the epidermis. Receptor-interacting protein kinase 4 (RIPK4) is an important regulator of keratinocyte differentiation, mutations of which are associated with congenital ectodermal malformations. In an attempt to identify the molecular basis of RIPK4's function, we applied yeast two-hybrid screen (Y2H) and found basal layer-specific keratin filament component keratin 14 (KRT14) as a novel RIPK4-interacting partner. During keratinocyte differentiation, layer-specific keratin composition is tightly regulated. Likewise, the basal layer specific KRT14/keratin 5 (KRT5) heterodimers are replaced by keratin 1 (KRT1)/keratin 10 (KRT10) in suprabasal layers. The regulation of keratin turnover is under the control of signaling associated with posttranslational modifications in which phosphorylation plays a major role. In this study, we verified the KRT14-RIPK4 interaction, which was identified with Y2H, in mammalian cells and showed that the interaction was direct by using proteins expressed in bacteria. According to our results, the N-terminal kinase domain of RIPK4 is responsible for KRT14-RIPK4 interaction; however, the RIPK4 kinase activity is dispensable for the interaction. In accordance with their interaction, RIPK4 and KRT14 colocalize within the cells, particularly at keratin filaments associated with perinuclear ring-like structures. Moreover, RIPK4 did not show any effect on KRT14/KRT5 heterodimer formation. Our results suggest that RIPK4 may regulate the keratin turnover required for keratinocyte differentiation through interacting with KRT14.