ubiquitin ligase Siah2


The Siah ubiquitin ligases are members of the RING finger E3 ligases. The Siah2 E3s are conserved from fly to mammals. Primarily implicated in cellular stress response, Siah ligases play a key role in hypoxia, through the regulation of HIF1a transcription, stability and activity. Concomitantly, physiological conditions associated with varying oxygen tension often highlight the importance of Siah, as seen in cancer and neurodegenerative disorders. Recent studies point to the importance of this ubiquitin ligase in tumor development and metastasis, as shown for melanoma and prostate cancer. Work from other groups has also demonstrated the importance of Siah2 in pancreatic, lung and breast tumor models.  Notably, Siah2 is involved in fundamental processes, including mitochondrial dynamics and tight junction.


Siah2 in prostate cancer  

Our studies in prostate cancer were fuled by the finding that cross of Siah KO mice (Siah2 homozygous KO x Siah1 heterozygous KO) with the TRAMP prostate cancer model, ehich drives the formation of neuroendocrine prostate tumors, resulted in attenuated tumor development, at the stage of hyperplasia. We found that reduced HIF1a level in the prostate tissues of these mice was sufficinet to attenuate transcriptional program that consisted of FOXa2 and HIF1a which otherwise drove neuroendocrine tumor formation. Further, we found that HES6, SOX9 and JMJD1a are the major downstream targets for the HIF1a/FOXa2 transcriptional program and that coexpression of these 3 was sufficient to restore the formation of neuroendocrine tumors. Furthermore, this mechanism was found to be as relevant for the neuroendocrine lesions (also known as NED or referred to prostate tumor stem cell niches) which are often found in adenocarcinoma of the prostate (~40%) and associate with the more mestatatic form of adenocarcinoma of the prostate and with poorer prognosis. 

In a followup study we have demonstrate that Siah2 is important for the formation of castration resistant prostate cancer (CRPC) also known as hormone refractory tumors. The mechanism underlying SIah1/2 contribution to CRPC entails the amplification of select pool of Androgen Receptor (AR) which elicits the increased expression of 15% of AR genes encoding for sterol biosynthesis parhway genes. Inhibition of Siah1/2 was shown effective in attenuating CRPC in mouse models.

These findings have prompted our efforts to develop inhibitors for SIah1/2. We developed a Siah1/2 inhibitory peptide which reached the phase of assessment in mouse models, where it is administered by i.v. injections, and are advancing the development of small molecule SIah1/2 inhibitors. 




 

Siah2 in melanoma 

The ubiquitin ligase Siah2 has been shown to regulate prolyl hydroxylase 3 (PHD3) stability with concomitant effect on HIF-1alpha availability. Because HIF-1alpha is implicated in tumorigenesis and metastasis, we used SW1 mouse melanoma cells, which develop primary tumors with a propensity to metastasize, in a syngeneic mouse model to assess a possible role for Siah2 in these processes. Inhibiting Siah2 activity by expressing a peptide designed to outcompete association of Siah2-interacting proteins reduced metastasis through HIF-1alpha without affecting tumorigenesis. Conversely, inhibiting Siah2 activity by means of a dominant-negative Siah2 RING mutant primarily reduced tumorigenesis through the action of Sprouty 2, a negative regulator of Ras signaling. Consistent with our findings, reduced expression of PHD3 and Sprouty2 was observed in more advanced stages of melanoma tumors. Using complementary approaches, these findings establish the role of Siah2 in tumorigenesis and metastasis by HIF-dependent and -independent mechanisms.


Siah2 in control of mitochondrial dynamics

Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. We have demonstrated that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.


Siah2 control of tight junction (TJ)

Changes in cell adhesion and polarity are closely associated with epithelial cell transformation and metastatic capacity. The tumor suppressor protein ASPP2 has been implicated in control of cell adhesion and polarity, through its effects on the PAR complex. In recent studies, we demonstrated that under hypoxic conditions the ubiquitin ligase Siah2 controls ASPP2 availability, with concomitant effect on epithelial cell polarity. LC-MS/MS analysis identified ASPP1 and ASPP2 as Siah2 interacting proteins. Biochemical analysis confirmed this interaction and mapped degron motifs within ASPP2, which are required for Siah2-medicated ubiquitination and proteasomal dependent degradation. Inhibition of Siah2 expression increases ASPP2 levels and enhances ASPP2-dependent mainenance of TJ integrity and polarized architecture in 3D organotypic culture. Conversly, increase of Siah2 expression under hypoxia decreases ASPP2 levels and the formation of apical polarity in 3D cultures. The finding of Sia2 regulation of TJ integrity and cell polarity through its regulation of ASPP2 stability points to unexpected mechanisms that can explain the impact of Siah2 on tumor development and metastasis.


Siah2 in the Unfolded Protein Response (UPR)

The search for mechanisms that activate Siah1/2 transcription led us to identify two of the three major UPR sensors as Siah1/2 transcriptional activators. ATF4 (along PERK) and sXBP1 (along IRE1) bind to respective response elements on Siah1/2 promoters and induces its transcription. Activation of Siah2 by the ATF4 and sXBP1 is stronger, compared with that of Siah1. Conditions required for ATF4 and sXBP1 activation of Siah1/2 transcription include severe stress conditions, including oxygen & glucose deprivation (mimicking ischemic conditions) or high levels of tunicamycin or tapsigargin. Once induced, Siah1/2 contribute to the amplification of ATF4 signaling, by degradation of PHD3, which limits ATF4 availability. In doing so, Siah1/2 contributes to the magnitude of the UPR signals, ampifying it under the more severe conditions. Under these conditions cells require Siah1/2 to undergo apoptosis. Inhibition of Siah1/2 would abrogate cell commitment to death programs, as demonstrated in vitro as in vivo in neuronal ischemia model. These findings establish Siah1/2 as an important component of the UPR - fine tuning the UPR per death or survival decitions. Since The regulation of cellular hypoxia is mediated also by Siah1/2 control of PHD3, Siah1/2 emerges as key regulator of fundamental cellular processes - the hypoxia and the UPR. 





OPEN QUESTIONS


Develop novel class inhibitors for Siah2 - using structure-based design and using thermal shift-based small molecule screens

Characterize novel pathways regulated by distinct Siah1 isoforms compared with Siah2.

Explore the role of Siah2 in neurodegenerative disorders, ischemia and cell metabolism





Supported by:


 

Hervey Family Fund @ San Diego Foundation. 

© Ronai Lab 2016