Notch-effector CSL promotes squamous cell carcinoma by repressing histone demethylase KDM6B
Dania Al Labban, Seung-Hee Jo, Paola Ostano, Chiara Saglietti, Massimo Bongiovanni, Renato Panizzon, G. Paolo Dotto
Dania Al Labban, Seung-Hee Jo, Paola Ostano, Chiara Saglietti, Massimo Bongiovanni, Renato Panizzon, G. Paolo Dotto
View: Text | PDF
Research Article Cell biology Oncology

Notch-effector CSL promotes squamous cell carcinoma by repressing histone demethylase KDM6B

Abstract

Notch 1/2 genes play tumor-suppressing functions in squamous cell carcinoma (SCC), a very common malignancy in skin and internal organs. In contrast with Notch, we show that the transcription factor CSL (also known as RBP-Jκ), a key effector of canonical Notch signaling endowed with intrinsic transcription-repressive functions, plays a tumor-promoting function in SCC development. Expression of this gene decreased in upper epidermal layers and human keratinocytes (HKCs) undergoing differentiation, while it increased in premalignant and malignant SCC lesions from skin, head/neck, and lung. Increased CSL levels enhanced the proliferative potential of HKCs and SCC cells, while silencing of CSL induced growth arrest and apoptosis. In vivo, SCC cells with increased CSL levels gave rise to rapidly expanding tumors, while cells with silenced CSL formed smaller and more differentiated tumors with enhanced inflammatory infiltrate. Global transcriptomic analysis of HKCs and SCC cells with silenced CSL revealed major modulation of apoptotic, cell-cycle, and proinflammatory genes. We also show that the histone demethylase KDM6B is a direct CSL-negative target, with inverse roles of CSL in HKC and SCC proliferative capacity, tumorigenesis, and tumor-associated inflammatory reaction. CSL/KDM6B protein expression could be used as a biomarker of SCC development and indicator of cancer treatment.

Authors

Dania Al Labban, Seung-Hee Jo, Paola Ostano, Chiara Saglietti, Massimo Bongiovanni, Renato Panizzon, G. Paolo Dotto

×

Figure 3

Positive role of CSL in promoting keratinocyte proliferative potential and cell survival.

Options: View larger image (or click on image) Download as PowerPoint
Positive role of CSL in promoting keratinocyte proliferative potential a...
(A) HKCs infected with retroviral vector for constitutive Myc-tagged CSL expression (mycCSL) in parallel with vector control (pmx), were analyzed for CSL expression by immunoblotting. Numbers refer to relative folds of CSL expression using GAPDH for normalization. (B) Three independent HKC strains, infected as in A, were tested for cell metabolic activity assays. Results are presented as luminescence intensity values relative to day 1. (C) The same HKC strains as in B were plated at limited density, and colony formation was measured. (D) HKCs infected with 2 shRNA lentiviruses against CSL (shCSL1 and shCSL2) versus empty control (PLKO1) were analyzed for CSL expression by immunoblotting. (E) Three HKC strains infected as in D were tested for cell metabolic assays. Lower luminescence/metabolic activity rates of control cells in panel B versus E are likely due to the use of cells with higher passage number in B, which was necessary because of lower efficiency of infection with PMX vectors used for those experiments. (F) The same HKC strains as in E assessed for colony formation. (G) The same HKC strains as in E were plated in Matrigel suspension cultures. The number of spheroids was counted using ImageJ software (NIH). Shown are representative images of spheroids formed by 1 HKC strain together with quantification of 3 strains. Scale bars: 250 μm. (H) The same HKC strains as in E were labeled with EdU for 6 hours. EdU-positive cells were counted using ImageJ software. Shown are representative images of cells of 1 HKC strain together with quantification of 3 strains. Scale bars: 100 μm. (B and C) Data are shown as mean ± SD, 2-tailed unpaired t test. n = 3 biological replicates/strain. (E–H) Data are shown as mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005, 1-way ANOVA with Dunnett’s test. n = 3 biological replicates/strain.