Background Infantile hemangioma (IH) is the most common benign tumor of infancy, yet its pathogenesis is poorly understood. Notch family members are known to play a role in vascular development during embryogenesis and postnatal tumor angiogenesis, yet the role of Notch signaling in the pathogenesis of IH has not been investigated. This study aims to survey Notch expression in IH. Materials & Methods RNA from resected hemangioma tissue and hemangioma-derived stem cells (HemSCs) and endothelial cells (HemECs) were used for gene expression analyses by real-time PCR. Results were confirmed with immunofluorescence (IF) for protein expression in tissue. Results Real-time PCR showed that Notch family gene expression in IH is distinct from placenta and skin. Notch3 is expressed in HemSCs, but not in HemECs, indicating Notch3 is downregulated as HemSCs differentiate into HemECs. Moreover, expression of endothelial-associated Notch proteins, Notch1, -4, and Jagged-1 are increased in involuting hemangiomas and HemECs, suggesting that as hemangioma progresses towards involution, it acquires more differentiated endothelium. A subset of cells stained double positive for Notch3 and CD31, pointing to a potential intermediate between the HemSC cellular differentiation into HemEC. Conclusion HemSCs have distinct Notch expression patterns from differentiated HemECs and from normal human endothelial cells. Notch3 is expressed in HemSCs while Notch1, Notch4, and Jagged-1 have higher expression levels in HemECs. Notch3 was localized to the interstitial cells outside of the nascent vascular channels in proliferating IH tissue sections, but became more apparent in the perivascular cells in involuting IH. In summary, the pattern of Notch gene expression mirrors the progression from immature cells to endothelial-lined vascular channels (i.e., endothelial differentiation) that characterizes the growth and involution of IH.

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After initial response to androgen receptor targeting drugs abiraterone or enzalutamide, most patients develop progressive disease and therefore, castration resistant prostate cancer (CRPC) remains a terminal disease. Multiple mechanisms underlying acquired resistance have been postulated. Intratumoral androgen synthesis may resume after abiraterone treatment. A point mutation in the ligand binding domain of androgen receptor may confer resistance to enzalutamide. Emergence of androgen receptor splice variants lacking the ligand binding domain may mediate resistance to abiraterone and enzalutamide. Steroid receptors such as glucocorticoid receptor may substitute for androgen receptor. Drugs with novel mechanisms of action or combination therapy, along with biomarkers for patient selection, may be needed to improve the therapy of CRPC.

Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

Aberrant activation of the androgen receptor (AR) may play a critical role in castration resistant prostate cancer. After ligand binding, AR is recruited to the androgen responsive element (ARE) sequences on the DNA where AR interaction with coactivators and corepressors modulates transcription. We demonstrated that phosphorylation of AR at Tyr-267 by Ack1/TNK2 tyrosine kinase results in nuclear translocation, DNA binding, and androgen-dependent gene transcription in a low androgen environment. In order to dissect downstream mechanisms, we searched for proteins whose interaction with AR was regulated by Ack1. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. Interaction between AR and SLIRP was disrupted by Ack1 kinase activity as well as androgen or heregulin treatment. The noncoding RNA, SRA, was required for AR-SLIRP interaction. SLIRP was bound to ARE’s of AR target genes in the absence of androgen. Treatment with androgen or heregulin led to dissociation of SLIRP from the ARE. Whole transcriptome analysis of SLIRP knockdown in androgen responsive LNCaP cells showed that SLIRP affects a significant subset of androgen-regulated genes. Our data suggest that Ack1 kinase and androgen regulate interaction between AR and SLIRP and that SLIRP functions as a coregulator of AR with properties of a corepressor in a context-dependent manner.