Suppression of pancreatic cancer growth and metastasis by HMP19 identified through genome-wide shRNA screen.

Therapeutic effectiveness against metastatic or even locally advanced pancreatic ductal adenocarcinoma (PDAC) is dismal, with 5-year survival less than 5%. Even in patients who undergo potentially curative resection, most patients' tumors recur in the liver. Improving therapies targeting or preventing liver metastases is crucial for improving prognosis. To identify genes suppressing metastasis, a genome-wide shRNA screen was done using the human non-metastatic PDAC cell line, S2-028. After identification of candidates, functional validation was done using intrasplenic and orthotopic injections in athymic mice. HMP19 strongly inhibited metastasis but also partially attenuated tumor growth in the pancreas. Knockdown of HMP19 increased localization of activated ERK1/2 in the nucleus, corresponding to facilitated cell proliferation, decreased p27(Kip1) and increased cyclin E1. Over-expression of HMP19 exerted the opposite effects. Using a tissue microarray of 84 human PDAC, patients with low expression of HMP19 showed significantly higher incidence of liver metastasis (p = 0.0175) and worse prognosis (p = 0.018) after surgery. HMP19, a new metastasis/tumor suppressor in PDAC, appears to alter signaling that leads to cell proliferation and appears to offer prognostic value in human PDAC.

Metastasis suppressors in breast cancers: mechanistic insights and clinical potential.

For the most part, normal epithelial cells do not disseminate to other parts of the body and proliferate, as do metastatic cells. Presumably, a class of molecules-termed metastasis suppressors-are involved in this homeostatic control. Metastasis suppressors are, by definition, cellular factors that, when re-expressed in metastatic cells, functionally inhibit metastasis without significantly inhibiting tumor growth. In this brief review, we catalog known metastasis suppressors, what is known about their mechanism(s) of action, and experimental and clinical associations to date.

A twin-cysteine motif in the V2 region of gp120 is associated with SIV envelope trimer stabilization.

The V1 and V2 variable regions of the primate immunodeficiency viruses contribute to the trimer association domain of the gp120 exterior envelope glycoprotein. A pair of V2 cysteine residues at 183 and 191 ("twin cysteines") is present in several simian immunodeficiency viruses, human immunodeficiency virus type 2 (HIV-2) and some SIV(cpz) lineages, but not in HIV-1. To examine the role of this potentially disulfide-bonded twin-cysteine motif, the cysteine residues in the SIVmac239 envelope glycoproteins were individually and pairwise substituted by alanine residues. All of the twin-cysteine mutants exhibited decreases in gp120 association with the Env trimer, membrane-fusing activity, and ability to support virus entry. Thus, the twin-cysteine motif plays a role in Env trimer stabilization in SIV and may do so in HIV-2 and some SIV(cpz) as well. This implies that HIV-1 lost the twin-cysteines, and may have relatively unstable Env trimers compared to SIV and HIV-2.

Human Ubc9 is involved in intracellular HIV-1 Env stability after trafficking out of the trans-Golgi network in a Gag dependent manner.

The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env) that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER) and its trafficking to the trans-Golgi network (TGN) were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.

Human Ubc9 contributes to production of fully infectious human immunodeficiency virus type 1 virions.

Ubc9 was identified as a cellular protein that interacts with the Gag protein of Mason-Pfizer monkey virus. We show here that Ubc9 also interacts with the human immunodeficiency virus type 1 (HIV-1) Gag protein and that their interaction is important for virus replication. Gag was found to colocalize with Ubc9 predominantly at perinuclear puncta. While cells in which Ubc9 expression was suppressed with RNA interference produced normal numbers of virions, these particles were 8- to 10-fold less infectious than those produced in the presence of Ubc9. The nature of this defect was assayed for dependence on Ubc9 during viral assembly, trafficking, and Env incorporation. The Gag-mediated assembly of virus particles and protease-mediated processing of Gag and Gag-Pol were unchanged in the absence of Ubc9. However, the stability of the cell-associated Env glycoprotein was decreased and Env incorporation into released virions was altered. Interestingly, overexpression of the Ubc9 trans-dominant-negative mutant C93A, which is a defective E2-SUMO-1 conjugase, suggests that this activity may not be required for interaction with Gag, virion assembly, or infectivity. This finding demonstrates that Ubc9 plays an important role in the production of infectious HIV-1 virions.

The pp24 phosphoprotein of Mason-Pfizer monkey virus contributes to viral genome packaging.

BACKGROUND: The Gag protein of Mason-Pfizer monkey virus, a betaretrovirus, contains a phosphoprotein that is cleaved into the Np24 protein and the phosphoprotein pp16/18 during virus maturation. Previous studies by Yasuda and Hunter (J. Virology. 1998. 72:4095-4103) have demonstrated that pp16/18 contains a viral late domain required for budding and that the Np24 protein plays a role during the virus life cycle since deletion of this N-terminal domain blocked virus replication. The function of the Np24 domain, however, is not known. RESULTS: Here we identify a region of basic residues (KKPKR) within the Np24 domain that is highly conserved among the phosphoproteins of various betaretroviruses. We show that this KKPKR motif is required for virus replication yet dispensable for procapsid assembly, membrane targeting, budding and release, particle maturation, or viral glycoprotein packaging. Additional experiments indicated that deletion of this motif reduced viral RNA packaging 6-8 fold and affected the transient association of Gag with nuclear pores. CONCLUSION: These results demonstrate that the Np24 domain plays an important role in RNA packaging and is in agreement with evidence that suggests that correct intracellular targeting of Gag to the nuclear compartment is an fundamental step in the retroviral life cycle.