Targeting PI3K and mTORC2 in metastatic renal cell carcinoma: new strategies for overcoming resistance to VEGFR and mTORC1 inhibitors.

With the advent of molecularly targeted agents, treatment of metastatic renal cell carcinoma (mRCC) has improved significantly. Agents targeting the vascular endothelial growth factor receptor (VEGFR) and the mammalian target of rapamycin complex 1 (mTORC1) are more effective and less toxic than previous standards of care involving cytotoxic and cytokine therapies. Unfortunately, many patients relapse following treatment with VEGFR and mTORC1 inhibitors as a result of acquired resistance mechanisms, which are thought to lead to the reestablishment of tumor vasculature. Specifically, the loss of negative feedback loops caused by inhibition of mTORC1 leads to upregulation of downstream effectors of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway and subsequent activation of hypoxia-inducible factor, an activator of angiogenesis. De novo resistance involving activated PI3K signaling has also been observed. These observations have led to the development of novel agents targeting PI3K, mTORC1/2 and PI3K/mTORC1/2, which have demonstrated antitumor activity in preclinical models of RCC. Several agents--BKM120, BEZ235 and GDC-0980--are being investigated in clinical trials in patients with metastatic/advanced RCC, and similar agents are being tested in patients with solid tumors. The future success of mRCC treatment will likely involve a combination of agents targeting the multiple pathways involved in angiogenesis, including VEGFR, PI3K and mTORC1/2.

Symplekin, a constitutive protein of karyo- and cytoplasmic particles involved in mRNA biogenesis in Xenopus laevis oocytes.

Symplekin is a dual location protein that has been localized to the cytoplasmic plaques of tight junctions but also occurs in the form of interchromatin particles in the karyoplasm. Here we report the identification of two novel and major symplekin-containing protein complexes in both the karyo- and the cytoplasm of Xenopus laevis oocytes. Buffer-extractable fractions from the karyoplasm of stage IV-VI oocytes contain an 11S particle, prepared by immunoselection and sucrose gradient centrifugation, in which symplekin is associated with the subunits of the cleavage and polyadenylation specificity factor (CPSF). Moreover, in immunofluorescence microscopy nuclear symplekin colocalizes with protein CPSF-100 in the "Cajal bodies." However, symplekin is also found in cytoplasmic extracts of enucleated oocytes and egg extracts, where it occurs in 11S as well as in ca. 65S particles, again in association with CPSF-100. This suggests that, in X. laevis oocytes, symplekin is possibly involved in both processes, 3'-end processing of pre-mRNA in the nucleus and regulated polyadenylation in the cytoplasm. We discuss the possible occurrence of similar symplekin-containing particles involved in mRNA metabolism in the nucleus and cytoplasm of other kinds of cells, also in comparison with the nuclear forms of other dual location proteins in nuclei and cell junctions.

Transcriptional activation of the general amino acid permease gene per1 by the histone deacetylase Clr6 Is regulated by Oca2 kinase.

Expression of nitrogen metabolism genes is regulated by the quality of the nitrogen supply. Here, we describe a mechanism for the transcriptional regulation of the general amino acid permease gene per1 in Schizosaccharomyces pombe. We show that when ammonia is used as the nitrogen source, low levels of per1 are transcribed and histones in the coding and surrounding regions of per1 are acetylated. In the presence of proline, per1 transcription is upregulated and initiates from a more upstream site, generating 5'-extended mRNAs. Concomitantly, histones at per1 are deacetylated in a Clr6-dependent manner, suggesting a positive role for Clr6 in transcriptional regulation of per1. Upstream initiation and histone deactylation of per1 are constitutive in cells lacking the serine/threonine kinase oca2, indicating that Oca2 is a repressor of per1. Oca2 interacts with a protein homologous to the Saccharomyces cerevisiae transcriptional activator Cha4 and with Ago1. Loss of Cha4 or Ago1 causes aberrant induction of per1 under noninducing conditions, suggesting that these proteins are also involved in per1 regulation and hence in nitrogen utilization.

[Genetic origin of non-syndromic cleft lip and palate. TWIST, a candidate gene? Research protocol]. / L'origine génétique des fentes labio-palatines non syndromiques. TWIST, gène candidat? Protocole de recherche.

Non syndromic cleft lip and palate (CLP) is the most frequent human malformation. CLP is of complex inheritance and at least twenty contributing chromosomal regions have been identified by linkage studies. On the other hand, mutations in several genes such as TWIST and FGFR2 result in syndromic cranio-facial abnormalities of highly variable range. It is our hypothesis that some mutations at TWIST might contribute to CLP in absence of other dysmorphic features. Thus, DNA biopsies of patients with non syndromic CLP are collected and prepared to search for allelic variations or mutations at TWIST. This study should contribute to improve the classification of facial malformations relative to gene, to help to a better understanding of the inheritance pattern of this pathology, to help to genetic counselling for some cases aiming at the prevention of genetic disease. This project is based on a close cooperation between the Orthodontic Department, the Paediatric Surgery Department and the Center for Clinical Investigation (University Hospital in Strasbourg), in a joint project with an academic research laboratory, expert in molecular biology and genetics.

Splicing factors stimulate polyadenylation via USEs at non-canonical 3' end formation signals.

The prothrombin (F2) 3' end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3' UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function. We show that the F2 USE contains a highly conserved nonameric core sequence, which promotes 3' end formation in a position- and sequence-dependent manner. We identify proteins that specifically interact with the USE, and demonstrate their function as trans-acting factors that promote 3' end formation. Interestingly, these include the splicing factors U2AF35, U2AF65 and hnRNPI. We show that these splicing factors not only modulate 3' end formation via the USEs contained in the F2 and the complement C2 mRNAs, but also in the biocomputationally identified BCL2L2, IVNS and ACTR mRNAs, suggesting a broader functional role. These data uncover a novel mechanism that functionally links the splicing and 3' end formation machineries of multiple cellular mRNAs in an USE-dependent manner.

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase.

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.