Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

HOXA1 Is an Antagonist of ERα in Breast Cancer.

Breast cancer is a heterogeneous disease and the leading cause of female cancer mortality worldwide. About 70% of breast cancers express ERα. HOX proteins are master regulators of embryo development which have emerged as being important players in oncogenesis. HOXA1 is one of them. Here, we present bioinformatic analyses of genome-wide mRNA expression profiles available in large public datasets of human breast cancer samples. We reveal an extremely strong opposite correlation between HOXA1 versus ER expression and that of 2,486 genes, thereby supporting a functional antagonism between HOXA1 and ERα. We also demonstrate in vitro that HOXA1 can inhibit ERα activity. This inhibition is at least bimodal, requiring an intact HOXA1 DNA-binding homeodomain and involving the DNA-binding independent capacity of HOXA1 to activate NF-κB. We provide evidence that the HOXA1-PBX interaction known to be critical for the transcriptional activity of HOXA1 is not involved in the ERα inhibition. Finally, we reveal that HOXA1 and ERα can physically interact but that this interaction is not essential for the HOXA1-mediated inhibition of ERα. Like other HOX oncoproteins interacting with ERα, HOXA1 could be involved in endocrine therapy resistance.

HOXA2 activity regulation by cytoplasmic relocation, protein stabilization and post-translational modification.

HOX proteins are homeodomain transcription factors critically involved in patterning animal embryos and controlling organogenesis. While the functions of HOX proteins and the processes under their control begin to be well documented, the modalities of HOX protein activity regulation remain poorly understood. Here we show that HOXA2 interacts with PPP1CB, a catalytic subunit of the Ser/Thr PP1 phosphatase complex. This interaction co-localizes in the cytoplasm with a previously described HOXA2 interactor, KPC2, which belongs to the KPC E3 ubiquitin ligase complex. We provide evidence that HOXA2, PPP1CB and KPC2 define a molecularly and functionally interacting complex. Collectively, our experiments support that PPP1CB and KPC2 together inhibit the activity of HOXA2 by activating its nuclear export, but favored HOXA2 de-ubiquitination and stabilization thereby establishing a store of HOXA2 in the cytoplasm.

Molecular Analysis of the HOXA2-Dependent Degradation of RCHY1.

The homeodomain transcription factor Hoxa2 interacts with the RING-finger type E3 ubiquitin ligase RCHY1 and induces its proteasomal degradation. In this work, we dissected this non-transcriptional activity of Hoxa2 at the molecular level. The Hoxa2-mediated decay of RCHY1 involves both the 19S and 20S proteasome complexes. It relies on both the Hoxa2 homeodomain and C-terminal moiety although no single deletion in the Hoxa2 sequence could disrupt the RCHY1 interaction. That the Hoxa2 homeodomain alone could mediate RCHY1 binding is consistent with the shared ability all the Hox proteins we tested to interact with RCHY1. Nonetheless, the ability to induce RCHY1 degradation although critically relying on the homeodomain is not common to all Hox proteins. This identifies the homeodomain as necessary but not sufficient for what appears to be an almost generic Hox protein activity. Finally we provide evidence that the Hoxa2-induced degradation of RCHY1 is evolutionarily conserved among vertebrates. These data therefore support the hypothesis that the molecular and functional interaction between Hox proteins and RCHY1 is an ancestral Hox property.

KPC2 relocalizes HOXA2 to the cytoplasm and decreases its transcriptional activity.

Regulation of transcription factor activity relies on molecular interactions or enzymatic modifications which influence their interaction with DNA cis-regulatory sequences, their transcriptional activation or repression, and stability or intracellular distribution of these proteins. Regarding the well-conserved Hox protein family, a restricted number of activity regulators have been highlighted thus far. In the framework of a proteome-wide screening aiming at identifying proteins interacting with Hoxa2, KPC2, an adapter protein constitutive of the KPC ubiquitin-ligase complex, was identified. In this work, KPC2 was confirmed as being a genuine interactor of Hoxa2 by co-precipitation and bimolecular fluorescence complementation assays. At functional level, KPC2 diminishes the transcriptional activity and induces the nuclear exit of Hoxa2. Gene expression analyses revealed that Kpc2 is active in restricted areas of the developing mouse embryo which overlap with the Hoxa2 expression domain. Together, our data support that KPC2 regulates Hoxa2 by promoting its relocation to the cytoplasm.