Insights into the tumor suppressor KCASH2: new functions and mechanisms of regulation

Medulloblastoma (MB) is the most common malignant childhood brain tumor. About 30% of all MBs belong to the I molecular subgroup, characterized by constitutive activation of the Sonic Hedgehog (Hh) pathway. The Hh pathway is involved in several fundamental processes during embryogenesis and in adult life and its deregulation may lead to cerebellar tumorigenesis. Indeed, Hh activity must be maintained via a complex network of activating and repressor signals. One of these repressor signals is KCASH2, belonging to the KCASH family of protein, which acts as negative regulators of the Hedgehog signaling pathway during cerebellar development and differentiation. KCASH proteins possess a well conserved N-terminal BTB domain but an heterogenous C-terminus, suggesting the presence of peculiar functions or different mechanisms of individual KCASHs functional regulation. In order to better characterize the physiologic role and modulation mechanisms of KCASH2, we have searched through a proteomic approach for new KCASH2 interactors, identifying Potassium Channel Tetramerization Domain Containing 15 (KCTD15) and Mitotic Arrest Deficient2-like 1 (MAD2). KCTD15 is able to directly interact with KCASH2, through its BTB/POZ domain. This interaction leads to increase KCASH2 stability which implies a reduction of the Hh pathway activity and a reduction of Hh-dependent MB cells proliferation. Here, we report the identification of KCTD15 as a novel player in the complex network of regulatory proteins which modulate Hh pathway, this could be a promising new target for therapeutic approach against MB. MAD2 is the main player in the spindle assembly complex (SAC), essential for chromosomal stability during cell mitosis, preventing defected cellular divisions that may lead to aneuploidy. Nowadays, no mechanisms have been provided for the MAD2 regulation, although it has been suggested that MAD2 may be degraded following ubiquitination by an unknown E3 ligase. Our work fills this gap, identifying in Cul3-KCASH2 the E3 ligase involved in MAD2 degradation process. Our data suggested that KCASH2 overexpression, affecting MAD2 protein levels, alters SAC formation during cell cycle, promoting mitotic defects that may give rise to chromosomal aberration and aneuploidy. The discovery of a mechanism able to modulate MAD2 protein levels and, indirectly, SAC checkpoint functionality and cell cycle progression may have important implications both in therapeutic approaches directed to the reconstitution of a normal SAC function and in approaches aiming to increase chromosomal instability to a level not sustainable by tumor cells, leading to their death. Finally, we have investigated the KCASH2 transcriptional regulation aimed to discover new potential therapeutics mechanisms for all that tumor types in which KCASH2 is low expressed. We have analyzed its proximal promoter region and performed bioinformatics analyses in order to identify putative transcription factors involved in KCASH2 regulation. Here, we have identified SP1, considered unanimously a hallmark of cancer, as a key transcriptional regulator that is involved in KCASH2 expression modulation in different cancers. The work presented here draw a more complex, although not yet complete, picture of the biological role of KCASH2, unveiling its additional functions as a new putative “guardian” of genomic stability and identifying two novel mechanism of regulation of its expression in KCTD15 and SP1.