The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.

A MYCN-independent mechanism mediating secretome reprogramming and metastasis in MYCN-amplified neuroblastoma.

MYCN amplification (MNA) is a defining feature of high-risk neuroblastoma (NB) and predicts poor prognosis. However, whether genes within or in close proximity to the MYCN amplicon also contribute to MNA+ NB remains poorly understood. Here, we identify that GREB1, a transcription factor encoding gene neighboring the MYCN locus, is frequently coexpressed with MYCN and promotes cell survival in MNA+ NB. GREB1 controls gene expression independently of MYCN, among which we uncover myosin 1B (MYO1B) as being highly expressed in MNA+ NB and, using a chick chorioallantoic membrane (CAM) model, as a crucial regulator of invasion and metastasis. Global secretome and proteome profiling further delineates MYO1B in regulating secretome reprogramming in MNA+ NB cells, and the cytokine MIF as an important pro-invasive and pro-metastatic mediator of MYO1B activity. Together, we have identified a putative GREB1-MYO1B-MIF axis as an unconventional mechanism promoting aggressive behavior in MNA+ NB and independently of MYCN.

HACE1 blocks HIF1α accumulation under hypoxia in a RAC1 dependent manner.

Uncovering the mechanisms that underpin how tumor cells adapt to microenvironmental stress is essential to better understand cancer progression. The HACE1 (HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase) gene is a tumor suppressor that inhibits the growth, invasive capacity, and metastasis of cancer cells. However, the direct regulatory pathways whereby HACE1 confers this tumor-suppressive effect remain to be fully elucidated. In this report, we establish a link between HACE1 and the major stress factor, hypoxia-inducible factor 1 alpha (HIF1α). We find that HACE1 blocks the accumulation of HIF1α during cellular hypoxia through decreased protein stability. This property is dependent on HACE1 E3 ligase activity and loss of Ras-related C3 botulinum toxin substrate 1 (RAC1), an established target of HACE1 mediated ubiquitinylation and degradation. In vivo, genetic deletion of Rac1 reversed the increased HIF1α expression observed in Hace1-/- mice in murine KRasG12D-driven lung tumors. An inverse relationship was observed between HACE1 and HIF1α levels in tumors compared to patient-matched normal kidney tissues, highlighting the potential pathophysiological significance of our findings. Together, our data uncover a previously unrecognized function for the HACE1 tumor suppressor in blocking HIF1α accumulation under hypoxia in a RAC1-dependent manner.

Development of Helicobacter pylori Whole-Proteome Arrays and Identification of Serologic Biomarkers for Noncardia Gastric Cancer in the MCC-Spain Study.

BACKGROUND: Helicobacter pylori (H. pylori) is a bacterial carcinogen and the leading risk factor for noncardia gastric cancer (NCGC). Detecting antibodies against specific H. pylori proteins in peripheral blood can be applied to characterize infection and determine disease associations. Most studies analyzing the association between H. pylori infection and gastric cancer have focused on previously identified antigens, predominantly the virulence factor cytotoxin-associated gene A (CagA). Selecting antigens in an unbiased approach may, however, allow the identification of novel biomarkers. METHODS: Using a combination of multiple spotting technique and cell-free, on-chip protein expression, we displayed the H. pylori genome (strain 26695) on high-density microarrays. Immunogenic proteins were identified by serum pool incubations and henceforth analyzed in individual samples. To test its applicability, we used sera from a multicase-control (MCC)-Spain study. Serologic responses between NCGC cases and controls were assessed by conditional logistic regression estimating ORs and 95% confidence intervals. RESULTS: We successfully expressed 93% of the 1,440 H. pylori open reading frames in situ. Of these, 231 (17%) were found to be immunogenic. By comparing 58 NCGC cases with 58 matched controls, we confirmed a higher seroprevalence of CagA among cases (66%) than controls (31%). We further identified a potential novel marker, the Helicobacter outer membrane protein A (HopA). CONCLUSIONS: In this study, we provide evidence that our H. pylori whole-proteome microarray offers a platform for unbiased de novo identification of serologic biomarkers. IMPACT: Given its versatile workflow, antibody responses against other H. pylori strains and possible associations with diverse H. pylori-related outcomes can be systematically analyzed.

HACE1 is a potential tumor suppressor in osteosarcoma.

Osteosarcoma is a malignant bone sarcoma characterized by extensive genomic disruption and a propensity for metastatic spread. Osteoid production suggests a close relationship with normal osteoblasts, and the latter are the presumptive cell of origin of this disease. The HACE1 gene, localized to human chromosome 6q21, encodes the HACE1 HECT E3 ligase, a tumor suppressor in diverse tumors that acts in part by targeting the activated form of RAC1 GTPase for proteasomal degradation. Disruption or loss of 6q21 is relatively common in osteosarcomas, and Hace1-/-/Tp53+/- mice frequently develop osteosarcomas, in contrast to Tp53+/- mice, which do not. This suggests an unexplored link between HACE1 loss and osteosarcoma. Here we compared HACE1 expression in normal osteoblasts and osteosarcoma cell lines in vitro by western blotting and quantitative RT-PCR, and in human osteosarcoma specimens by immunohistochemistry. Both HACE1 transcript and protein levels were reduced in osteosarcoma compared to osteoblasts in vitro. Reduced HACE1 expression in osteosarcoma tumors was observed in 76% of cases and associated with high-grade lesions. Further, clonally derived pairs of high and low metastatic osteosarcoma cell lines showed significant downregulation in the high compared to corresponding low metastatic cells. Ectopic expression of HACE1 markedly inhibited anchorage-independent growth and cell motility of HACE1 osteosarcoma cell lines, and was associated with reduced RAC1 activation and decreased reactive oxygen species (ROS). Finally, HACE1 overexpression blocked osteosarcoma xenograft growth and dramatically reduced pulmonary metastases. These findings point to a potential tumor suppressor function for HACE1 in osteosarcoma.