Abstract
The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5+ malignant melanoma-initiating cells (ABCB5+ MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK–STAT (janus kinase–signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK–STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro. Finally, using a stem cell proliferation assay and tumour xenotransplantation assay in non-obese diabetic/severe combined immunodeficiency mice, we show that HAGE promotes MMICs-dependent tumour initiation and tumour growth by preventing the anti-proliferative effects of interferon-α (IFNα). Our results suggest that the helicase HAGE has a key role in the resistance of ABCB5+ MMICs to IFNα treatment and that cancer therapies targeting HAGE may have broad implications for the treatment of malignant melanoma.
Highlights
IFNa signals through the JAK–STAT pathway and results in the induction of several genes
To investigate the expression status of PML in HAGE þ and ATP-binding cassette member 5 (ABCB5) þ melanoma-initiating cells (MMICs), we performed immunostaining on a malignant melanoma tissue microarray (TMA) using antibodies against PML, HAGE and ABCB5
PML expression was significantly decreased in HAGE þ and ABCB5 þ tumour cells when compared with the control
Summary
IFNa signals through the JAK–STAT (janus kinase–signal transducers and activators of transcription) pathway and results in the induction of several genes This endows IFNa with multiple effects in a variety of malignancies that range from antiangiogenic effects to potent immunoregulatory, differentiation-inducing, pro-apoptotic and anti-proliferative properties.[8,9] The gene encoding promyelocytic leukaemia protein (PML) is a well-known downstream target of IFNs signalling and its induction by IFNs results in a significant increase in the expression of PML and the number of PML nuclear bodies (PML-NBs).[10,11,12] PML, a member of the Ring-. An in vitro unwinding assay provides a mechanistic insight by demonstrating the capacity of the helicase HAGE to unwind SOCS1 RNA complexes and thereby promote the expression of SOCS1 protein These findings support the model by which HAGE promotes the initiation of tumours by ABCB5 þ MMICs and their resistance to the anti-proliferative effects of IFNa by inactivating the JAK–STAT pathway which is necessary for PML expression via a mechanism which involves the SOCS1
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