An effective AKT inhibitor-PARP inhibitor combination therapy for recurrent ovarian cancer.

BACKGROUND: Although the use of PARP inhibitor has received considerable amount of attention in ovarian cancer, PARP inhibitor resistance still emerges with disease progression. PI3K/AKT pathway inhibitors have been proposed to synergize with PARP inhibition to slow tumor growth, but the exact molecular mechanisms are still elusive. METHODS: Utilizing tumor samples from recurrent EOC patients with platinum resistance and prior PARP inhibitor use, Mini PDX and PDX models were established to study the anti-tumor effect of AKT inhibitor (LAE003) and LAE003/PARP inhibitor (Olaparib) in combination. Five ovarian cancer cell lines were treated with Olaparib or LAE003 or in combination in vitro. Cell viability and apoptosis rate were measured after the treatments. Combination index by the Chou-Talalay was used to evaluate in vitro combination effect of Olaparib and LAE003. The protein expression level of PARP1 and PAR was measured by Western blot in cell lines and by immunohistochemistry in PDX tumor tissues. RESULTS: Tumor cells from two out of five platinum-resistant ovarian cancer patients previously treated with PARP inhibitor were sensitive to AKT inhibition in Mini-PDX study. Inhibition of AKT further increased the response of tumor cells to Olaparib in a PDX model derived from a recurrent platinum-resistant ovarian cancer patient. Additive anti-proliferation effect of LAE003 and Olaparib was also observed in three ovarian cancer cell lines with high PARP1 protein level. Interestingly, mechanism study revealed that AKT inhibition decreased PARP enzyme activity as measured by PAR level and/or reduced PARP1 protein level in the tumor cell lines and PDX tumor tissues, which may explain the observed combined anti-tumor effect of LAE003 and Olaparib. CONCLUSION: Collectively, our results suggest that the combination of AKT inhibitor and PARP inhibitor could be a viable approach for clinical testing in recurrent ovarian cancer patients.

Circulating Cell-Free DNA-Based Detection of Tumor Suppressor Gene Copy Number Loss and Its Clinical Implication in Metastatic Prostate Cancer.

Current liquid biopsy assays lack sufficient sensitivity to detect copy number loss, which limits the interrogation of critical tumor suppressor gene deletions during cancer progression and treatment. Here we describe a liquid biopsy assay with improved sensitivity for detection of copy number loss in blood samples with low levels of circulating tumor DNA, and demonstrate its utility by profiling PTEN, RB1, and TP53 genetic loss in metastatic prostate cancer patients.

Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-ß signalling.

In advanced cancers, the TGF-ß pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-ß signalling. NR4A1 promotes TGF-ß/SMAD signalling by facilitating AXIN2-RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-ß-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-ß-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-ß-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-ß signalling in breast cancer.

TRAF4 promotes TGF-ß receptor signaling and drives breast cancer metastasis.

TGF-ß signaling is a therapeutic target in advanced cancers. We identified tumor necrosis factor receptor-associated factor 4 (TRAF4) as a key component mediating pro-oncogenic TGF-ß-induced SMAD and non-SMAD signaling. Upon TGF-ß stimulation, TRAF4 is recruited to the active TGF-ß receptor complex, where it antagonizes E3 ligase SMURF2 and facilitates the recruitment of deubiquitinase USP15 to the TGF-ß type I receptor (TßRI). Both processes contribute to TßRI stabilization on the plasma membrane and thereby enhance TGF-ß signaling. In addition, the TGF-ß receptor-TRAF4 interaction triggers Lys 63-linked TRAF4 polyubiquitylation and subsequent activation of the TGF-ß-activated kinase (TAK)1. TRAF4 is required for efficient TGF-ß-induced migration, epithelial-to-mesenchymal transition, and breast cancer metastasis. Elevated TRAF4 expression correlated with increased levels of phosphorylated SMAD2 and phosphorylated TAK1 as well as poor prognosis among breast cancer patients. Our results demonstrate that TRAF4 can regulate the TGF-ß pathway and is a key determinant in breast cancer pathogenesis.

Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6.

SMAD6 is a crucial feedback inhibitory regulator of bone morphogenetic protein (BMP)/SMAD signalling. Although little is known regarding the post-transcriptional modification of inhibitory SMADs and the mechanism by which their function is regulated. In this study, using a whole proteomic interaction screen for SMAD6, we identified a large putative E2 ubiquitin-conjugating enzyme UBE2O (E2-230K) as a novel interacting protein of SMAD6. We showed that UBE2O functions as an E2-E3 hybrid to monoubiquitinate SMAD6 at lysine 174 and that the cysteine 885 residue of human UBE2O is necessary for SMAD6 monoubiquitination. Inactivation of the SMAD6 monoubiquitination site specially potentiates the inhibitory ability of SMAD6 against BMP7-induced SMAD1 phosphorylation and transcriptional responses. We also found that UBE2O potentiated BMP7 signalling in a SMAD6-dependent manner. Addressing the molecular mechanism by which UBE2O and monoubiquitinated SMAD6 potentiate BMP7 signalling, we demonstrated that monoubiquitinated SMAD6 impairs the binding affinity of non-modified SMAD6 to the BMP type I receptor. Moreover, UBE2O and SMAD6 cooperated in the regulation of BMP7-induced adipogenesis.

Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation.

Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.

Rescue screens with secreted proteins reveal compensatory potential of receptor tyrosine kinases in driving cancer growth.

The overall power of kinase inhibitors is substantially overshadowed by the acquisition of drug resistance. To address this issue, we systematically assessed the potential of secreted proteins to induce resistance to kinase inhibitors. To this end, we developed a high-throughput platform for screening a cDNA library encoding 3,432 secreted proteins in cellular assays. Using cancer cells originally dependent on either MET, FGFR2, or FGFR3, we observed a bypass of dependence through ligand-mediated activation of alternative receptor tyrosine kinases (RTK). Our findings indicate a broad and versatile potential for RTKs from the HER and FGFR families as well as MET to compensate for loss of each other. We further provide evidence that combined inhibition of simultaneously active RTKs can lead to an added anticancer effect.

USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-ß type I receptor.

The stability and membrane localization of the transforming growth factor-ß (TGF-ß) type I receptor (TßRI) determines the levels of TGF-ß signalling. TßRI is targeted for ubiquitylation-mediated degradation by the SMAD7-SMURF2 complex. Here we performed a genome-wide gain-of-function screen and identified ubiquitin-specific protease (USP) 4 as a strong inducer of TGF-ß signalling. USP4 was found to directly interact with TßRI and act as a deubiquitylating enzyme, thereby controlling TßRI levels at the plasma membrane. Depletion of USP4 mitigates TGF-ß-induced epithelial to mesenchymal transition and metastasis. Importantly, AKT (also known as protein kinase B), which has been associated with poor prognosis in breast cancer, directly associates with and phosphorylates USP4. AKT-mediated phosphorylation relocates nuclear USP4 to the cytoplasm and membrane and is required for maintaining its protein stability. Moreover, AKT-induced breast cancer cell migration was inhibited by USP4 depletion and TßRI kinase inhibition. Our results uncover USP4 as an important determinant for crosstalk between TGF-ß and AKT signalling pathways.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

TGF-ß members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-ß family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-ß signaling. Depletion of RNF12 dramatically reduced TGF-ß/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-ß family signaling.

Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.

Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/ß-catenin signaling. We found the extracellular ß-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.