Cancer remains a leading cause of human death. During the development of cancer, growth related signaling pathways are continuously activated. Sustaining cell proliferation is thus considered one of six hallmarks in cancer. In dense populations of normal cells, the growth signaling could be diminished in response to cell-cell contact, however, whose inhibition is abolished in various types of cancer cells. Until recently, the mechanism basis for evading contact inhibition remains elusive. As ackonwledged, PI3K-AKT, MAPK-ERK and Hippo-YAP pathways are highly involved in maintaining fast growth of cancer cells. Recent studies indicated that these three pathways also contribute to contact inhibition in densely cultured cells. CD44 is a ubiquitously expressed glycoprotein that functions in cell-cell communication. However, the function of CD44 in contact inhibition，maintenance of cancer cell stemness and how CD44 integrating multiple pathways have yet to be fully established. Herein, this work further explored the signaling networks regulated by CD44 and its role in contact inhibition evasion, cell growth, cell cycle as well as maintenance of stemness.
In the present study, western blot results indicated that high-dense culture of MDA-MB-435S cells would lead to activation of Hippo-YAP pathway with an increase of p-YAP/YAP ratio. In addition, the phosphorylation level of AKT and ERK1/2 were also elevated, indicating an abnormal activation of PI3K-AKT and MAPK-ERK signaling pathways towards contact inhibition. To explore the role of CD44 in contact inhibition, protein levels of AKT, ERK1/2, YAP and their phosphorylation status were measured in MDA-MB-435S and BT549 cells which treated with CD44 siRNA. Decrease of AKT, ERK1/2 phosphorylation level and increase of phospho-YAP level were observed indicating a common upstream regulator of CD44 to these three pathways. Importantly, NF2, a well-known contact inhibition mediator and CD44 interacting protein, was activated in response to CD44 silencing. The phosphorylation level of LATS1/2, a core component of Hippo-YAP pathway, was dropped in CD44-silenced MDA-MB-435S cells. These data suggested that CD44 participated in evading contact inhibition of cancer cells via regulating a network including MAPK-ERK pathway, PI3K-AKT pathway and Hippo-YAP pathway. To further evaluate the interplay among PI3K-AKT, MAPK-ERK and Hippo-YAP pathway, protein levels of core components of these pathways were detected in cells treated with vehicle or LY294002 (PI3K inhibitor) or MK2206 (AKT inhibitor) or PD0325901 (MAPK inhibitor). Inhibition of MAPK-ERK pathway would result in an compensatory activation of PI3K-AKT pathway. The interplay between MAPK-ERK pathway and Hippo-YAP pathway was far more complex, while siRNA mediated knockdown of ERK1 induced an increase of YAP protein level through transcriptional activation, ERK2 silencing destablized YAP to reduce YAP protein level. Moreover, transcription level of ERK1 was negatively correlated with YAP and 11 target genes of YAP in transcriptome of 51 breast cancer cell lines analyzed. Additionally, higher ERK1 expression was associated with better clinical outcome in breast cancer patients. Finally, cell behaviors in cells treated with CD44 siRNA was studied. Silencing of CD44 would generate changes as followed: 1. inhibition of cell proliferation with a decrease of PCNA mRNA level. 2. cell cycle perturbation with prolonged G2/M phase and shortened G1 phase, elevated transcription of key G1/S checkpoint protein Cyclin D1 and Cyclin E2, transcriptional inactivation of CDK1. 3. reduced cancer cell stemness with a reduced size/number of manmosphere, tumor volume of nude mice, plate colony number, KLF4 protein and mRNA levels.
In summary, this study indicated that abnormal activation of MAPK-ERK pathway and PI3K-AKT pathway contribute to contact inhibition evasion in cancer cells. During contact inhibition, CD44 functions as an upstream regulator to activate PI3K-AKT pathway, MAPK-ERK pathway and inhibit Hippo-YAP pathway. The regulation of Hippo-YAP pathway by CD44 was realized via interaction with NF2. In addition, crosstalk amongst PI3K-AKT pathway, MAPK-ERK pathway and Hippo-YAP pathway was discovered in cancer cells. Inhibition of MAPK-ERK pathway was accompanied by activation of PI3K-AKT pathway. Importantly, ERK1 and ERK2, core kinases of MAPK-ERK pathway, regulated YAP in two different mechanisms that have not been discovered before. ERK1 repressed YAP transcription and ERK2 protected YAP from degradation. Additionally, as a common upstream regulator, CD44 participated in regulating cell proliferation, cell cycle and cancer cell stemness through transcriptional regulation of key genes in these processes.
This study not only discovered the molecular mechanism of CD44 during cancer development, but also facilitated further improvement of cancer therapy.