Notch-effector CSL promotes squamous cell carcinoma by repressing histone demethylase KDM6B
Dania Al Labban, … , Renato Panizzon, G. Paolo Dotto
Dania Al Labban, … , Renato Panizzon, G. Paolo Dotto
Published May 14, 2018
Citation Information: J Clin Invest. 2018;128(6):2581-2599. https://doi.org/10.1172/JCI96915.
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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

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Figure 10

KDM6B expression increases in HKCs and SCC cells with CSL gene silencing and decreases in neoplastic lesions.

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KDM6B expression increases in HKCs and SCC cells with CSL gene silencing...
(A) Three independent HKC strains infected with 2 shRNA lentiviruses against CSL were analyzed by RT-qPCR for KDM6B mRNA expression with 36B4 for normalization; 2-tailed 1-sample t test. n = 3 HKC strains. (B) Indicated SCC cell lines infected with 2 shRNA lentiviruses against CSL were analyzed by RT-qPCR for KDM6B mRNA expression with 36B4 for normalization. Data are shown as mean ± SEM, 2-tailed 1-sample t test. n = 3 independent experiments. (C) Immunofluorescence analysis of KDM6B expression in HKCs and SCC13 and SCCO22 cells with CSL gene silencing, with DAPI staining for cell identification. Shown are representative images of HKC staining (left panel) and quantification of fluorescence signal using ImageJ software from all tested cell lines (right panel). Data are shown as mean ± SEM, 2-tailed 1-sample t test. Scale bars: 150 μm. (D) The same RNA samples as in Figure 8C were examined by RT-qPCR for KDM6B expression. Data are shown as mean ± SEM, 1-tailed 1-sample t test. n = 4 mice for SCC13 at week 1; n = 5 mice for SCC13 at week 2; n = 5 mice for SCCO28 at week 1; n = 4 mice for SCCO28 at week 2. (E) LCM-obtained epidermis from AK and skin SCC lesions versus flanking normal skin were analyzed by RT-qPCR for KDM6B expression. P, patients. n = 6 AK regions; n = 6 normal regions; n = 2 SCC regions; n = 2 normal regions. (A–E) *P < 0.05; **P < 0.005; ***P < 0.0005, 2-tailed paired t test.