EXOC4 Promotes Diffuse-Type Gastric Cancer Metastasis via Activating FAK Signal.

In comparison with intestinal-type gastric cancer, diffuse-type gastric cancer (DGC) is more likely to recur, metastasize, and exhibit worse clinical outcomes; however, the underlying mechanism of DGC recurrence remains elusive. By employing an LC/MS-MS proteomic approach, we identified that exocyst complex component 4 (EXOC4) was significantly upregulated in DGC with recurrence, compared to those with nonrecurrence. High expression of EXOC4 was correlated with tumor metastasis and poor prognosis in patients with DGC. Moreover, EXOC4 promoted cell migration and invasion as well as the tumor metastasis of DGC cells. Mechanistically, EXOC4 regulated the phosphorylation of focal adhesion kinase (FAK) at Y397 sites by stimulating the secretion of integrin α5/ß1/EGF and enhancing the interaction of FAK and integrin or EGFR. The FAK inhibitor VS-4718 reversed the metastasis mediated by EXOC4 overexpression and suppressed the tumor growth of patient-derived xenografts derived from DGC with high EXOC4 expression. The EXOC4-FAK axis could be a potential therapeutic target for patients with DGC with high expression of EXOC4. IMPLICATIONS: The EXOC4-FAK axis promoted DGC metastasis and could be a potential therapeutic target for patients with DGC.

OTUD6A promotes prostate tumorigenesis via deubiquitinating Brg1 and AR.

Ovarian tumor (OTU) subfamily deubiquitinases are involved in various cellular processes, such as inflammation, ferroptosis and tumorigenesis; however, their pathological roles in prostate cancer (PCa) remain largely unexplored. In this study, we observed that several OTU members displayed genomic amplification in PCa, among which ovarian tumor deubiquitinase 6A (OTUD6A) amplified in the top around 15-20%. Further clinical investigation showed that the OTUD6A protein was highly expressed in prostate tumors, and increased OTUD6A expression correlated with a higher biochemical recurrence risk after prostatectomy. Biologically, wild-type but not a catalytically inactive mutant form of OTUD6A was required for PCa cell progression. In vivo experiments demonstrated that OTUD6A oligonucleotides markedly suppressed prostate tumorigenesis in PtenPC-/- mice and patient-derived xenograft (PDX) models. Mechanistically, the SWI/SNF ATPase subunit Brg1 and the nuclear receptor AR (androgen receptor) were identified as essential substrates for OTUD6A in PCa cells by a mass spectrometry (MS) screening approach. Furthermore, OTUD6A stabilized these two proteins by erasing the K27-linked polyubiquitination of Brg1 and K11-linked polyubiquitination of AR. OTUD6A amplification exhibited strong mutual exclusivity with mutations in the tumor suppressors FBXW7 and SPOP. Collectively, our results indicate the therapeutic potential of targeting OTUD6A as a deubiquitinase of Brg1 and AR for PCa treatment.

5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma.

Approximately 20% of multiple myeloma (MM) are caused by a chromosomal translocation t (4; 14) that leads to the overexpression of the nuclear receptor binding SET domain-protein 2 (NSD2) histone methyltransferase. NSD2 catalyzes the methylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Using high-throughput screening (HTS) with biological analyses, a series of 5-aminonaphthalene derivatives were designed and synthesized as novel NSD2 inhibitors. Among all the prepared compounds, 9c displayed a good NSD2 inhibitory activity (IC50 = 2.7 µM) and selectivity against both SET-domain-containing and non-SET-domain-containing methyltransferases. Preliminary research indicates the inhibition mechanism of compound 9c by significantly suppressed the methylation of H3K36me2. Compound 9c specifically inhibits the proliferation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 by inducing cell cycle arrest and apoptosis with little cytotoxicity. It has been reported that the anti-cancer effect of compound 9c is partly achieved by completely suppressing the transcriptional activation of NSD2-targeted genes. When administered intraperitoneally at 25 mg/kg, compound 9c suppressed the tumor growth of RS4:11 xenografts in vivo and no body weight loss was detected in the tested SCID mice.

G3BP1 interacts with YWHAZ to regulate chemoresistance and predict adjuvant chemotherapy benefit in gastric cancer.

BACKGROUND: A large proportion of gastric cancer patients are susceptible to chemoresistance, while the underlying mechanism remains obscure. Stress granules (SGs) play a self-defence role for tumour cells in inhibiting chemotherapy-induced apoptosis. As an SG assembly effector, G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein) has been reported to be overexpressed in gastric cancer; thus, here we aim to explore its potent roles in gastric cancer chemoresistance. METHODS: Kaplan-Meier analysis was used to compare survival rates in gastric cancer patients with different G3BP1 expression. The influence of G3BP1 on gastric cancer cell chemoresistance and apoptosis were evaluated by in vitro and in vivo approaches. The interaction between G3BP1 and YWHAZ was assessed by immunohistochemistry, immunoprecipitation and immunofluorescence. RESULTS: G3BP1 was associated with the poor outcome of gastric cancer patients who received adjuvant chemotherapy. G3BP1 knockdown significantly increased the sensitivity of gastric cancer cells to chemotherapy drugs. Mechanically, cell apoptosis and pro-apoptotic-associated molecules were significantly elevated upon G3BP1 depletion. Gene co-expression network analyses identified YWHAZ as the critical interlayer of G3BP1; as a result, G3BP1 interacted with YWHAZ to sequester Bax into the cytoplasm. Clinically, G3BP1highYWHAZhigh gastric cancer patients displayed the worst outcome compared with other patients after chemotherapy. CONCLUSIONS: The expression of G3BP1 and YWHAZ could predict the adjuvant chemotherapy benefit in gastric cancer patients.

BBB-penetrating codelivery liposomes treat brain metastasis of non-small cell lung cancer with EGFRT790M mutation.

EGFR TKI therapy has become a first-line regimen for non-small cell lung cancer (NSCLC) patients with EGRF mutations. However, there are two big challenges against effective therapy--the secondary EGFR mutation-associated TKI resistance and brain metastasis (BMs) of lung cancer. The BMs is a major cause of death for advanced NSCLC patients, and the treatment of BMs with TKI resistance remains difficult. Methods: Tumor-associated macrophages (TAM) is a promising drug target for inhibiting tumor growth, overcoming drug resistance, and anti-metastasis. TAM also plays an essential role in regulating tumor microenvironment. We developed a dual-targeting liposomal system with modification of anti-PD-L1 nanobody and transferrin receptor (TfR)-binding peptide T12 for codelivery of simvastatin/gefitinib to treat BMs of NSCLC. Results: The dual-targeting liposomes could efficiently penetrate the blood-brain barrier (BBB) and enter the BMs, acting on TAM repolarization and reversal of EGFRT790M-associated drug resistance. The treatment mechanisms were related to the elevating ROS and the suppression of the EGFR/Akt/Erk signaling pathway. Conclusion: The dual-targeting liposomal codelivery system offers a promising strategy for treating the advanced EGFRT790M NSCLC patients with BMs.

CUDC-907 displays potent antitumor activity against human pancreatic adenocarcinoma in vitro and in vivo through inhibition of HDAC6 to downregulate c-Myc expression.

Pancreatic adenocarcinoma is a highly malignant cancer that often involves a deregulation of c-Myc. It has been shown that c-Myc plays a pivotal role in the regulation of a variety of physiological processes and is involved in early neoplastic development, resulting in poor progression. Hence, suppression of c-Myc overexpression is a potential strategy for pancreatic cancer therapy. CUDC-907 is a novel dual-acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC). It has shown potential efficiency in patients with lymphoma, multiple myeloma, or thyroid cancer, as well as in solid tumors with c-Myc alterations, but the evidence is lacking for how CUDC-907 regulates c-Myc. In this study, we investigated the effect of CUDC-907 on human pancreatic cancer cells in vitro and in vivo. Our results showed that CUDC-907 potently inhibited the proliferation of 9 pancreatic cancer cell lines in vitro with IC50 values ranging from 6.7 to 54.5 nM. Furthermore, we revealed the antitumor mechanism of CUDC-907 in Aspc-1, PANC-1, and Capan-1 pancreatic cancer cells: it suppressed the HDAC6 subunit, thus downregulating c-Myc protein levels, which was a mode of action distinct from the existing mechanisms. Consistently, the extraordinary antitumor activity of CUDC-907 accompanied by downregulation of c-Myc and Ki67 expression in tumor tissue was observed in a human pancreatic cancer Aspc-1 xenograft nude mouse model in vivo. Our results suggest that CUDC-907 can be a valuable therapeutic option for treating pancreatic adenocarcinoma.

Remodeling Tumor-Associated Macrophages and Neovascularization Overcomes EGFRT790M -Associated Drug Resistance by PD-L1 Nanobody-Mediated Codelivery.

Precision medicine has made a significant breakthrough in the past decade. The most representative success is the molecular targeting therapy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in non-small-cell lung cancer (NSCLC) with oncogenic drivers, approved by the US Food and Drug Administration (FDA) as first-line therapeutics for substituting chemotherapy. However, the rapidly developed TKI resistance invariably leads to unsustainable treatment. For example, gefitinib is the first choice for advanced NSCLC with EGFR mutation, but most patients would soon develop secondary EGFRT790M mutation and acquire gefitinib resistance. TKI resistance is a severe emergency issue to be solved in NSCLC, but there are a few investigations of nanomedicine reported to address this pressing problem. To overcome EGFRT790M -associated drug resistance, a novel delivery and therapeutic strategy is developed. A PD-L1 nanobody is identified, and first used as a targeting ligand for liposomal codelivery. It is found that simvastatin/gefitinib combination nanomedicine can remodel the tumor microenvironment (e.g., neovascularization regulation, M2-macrophage repolarization, and innate immunity), and display the effectiveness of reversing the gefitinib resistance and enhancing the EGFRT790M -mutated NSCLC treatment outcomes. The novel simvastatin-based nanomedicine provides a clinically translatable strategy for tackling the major problem in NSCLC treatment and demonstrates the promise of an old drug for new application.

SCFFBW7-mediated degradation of Brg1 suppresses gastric cancer metastasis.

Brg1/SMARCA4 serves as the ATPase and the helicase catalytic subunit for the multi-component SWI/SNF chromatin remodeling complex, which plays a pivotal role in governing chromatin structure and gene transcription. However, the upstream signaling pathways regulating Brg1 protein stability and its physiological contribution to carcinogenesis remain largely elusive. Here we report that Brg1 is a bona fide ubiquitin substrate of SCFFBW7. We reveal that CK1δ phosphorylates Brg1 at Ser31/Ser35 residues to facilitate the binding of Brg1 to FBW7, leading to ubiquitination-mediated degradation. In keeping with a tumor suppressive role of FBW7 in human gastric cancer, we find an inverse correlation between FBW7 and Brg1 expression in human gastric cancer clinical samples. Mechanistically, we find that stabilization of Brg1 in gastric cancer cells suppresses E-cadherin expression, subsequently promoting gastric cancer metastasis. Hence, this previously unknown FBW7/Brg1 signaling axis provides the molecular basis and the rationale to target Brg1 in FBW7-compromised human gastric cancers.

Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors.

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.

Piribedil disrupts the MLL1-WDR5 interaction and sensitizes MLL-rearranged acute myeloid leukemia (AML) to doxorubicin-induced apoptosis.

Targeting WT MLL for the treatment of MLL-r leukemia, which is highly aggressive and resistant to chemotherapy, has been shown to be a promising strategy. However, drug treatments targeting WT MLL are lacking. We used an in vitro histone methyltransferase assay to screen a library consists of 592 FDA-approved drugs for MLL1 inhibitors by measuring alterations in HTRF signal and found that Piribedil represented a potent activity. Piribedil specifically inhibited the proliferation of MLL-r cells by inducing cell-cycle arrest, apoptosis and myeloid differentiation with little toxicity to the non-MLL cells. Mechanism study showed Piribedil blocked the MLL1-WDR5 interaction and thus selectively reduced MLL1-dependent H3K4 methylation. Importantly, MLL1 depletion induced gene expression that was similar to that induced by Piribedil and rendered the MLL-r cells resistant to Piribedil-induced toxicity, revealing Piribedil exerted anti-leukemia effects by targeting MLL1. Furthermore, both the Piribedil treatment and MLL1 depletion sensitized the MLL-r cells to doxorubicin-induced apoptosis. Our study support the hypothesis that Piribedil could serve as a new drug for the treatment of MLL-r AML and provide new insight for further optimization of targeting MLL1 HMT activity.

CTC clusters induced by heparanase enhance breast cancer metastasis.

Aggregated metastatic cancer cells, referred to as circulating tumor cell (CTC) clusters, are present in the blood of cancer patients and contribute to cancer metastasis. However, the origin of CTC clusters, especially intravascular aggregates, remains unknown. Here, we employ suspension culture methods to mimic CTC cluster formation in the circulation of breast cancer patients. CTC clusters generated using these methods exhibited an increased metastatic potential that was defined by the overexpression of heparanase (HPSE). Heparanase induced FAK- and ICAM-1-dependent cell adhesion, which promoted intravascular cell aggregation. Moreover, knockdown of heparanase or inhibition of its activity with JG6, a heparanase inhibitor, was sufficient to block the formation of cell clusters and suppress breast cancer metastasis. Our data reveal that heparanase-mediated cell adhesion is critical for metastasis mediated by intravascular CTC clusters. We also suggest that targeting the function of heparanase in cancer cell dissemination might limit metastatic progression.

Aspirin disrupts the mTOR-Raptor complex and potentiates the anti-cancer activities of sorafenib via mTORC1 inhibition.

Aspirin is associated with a reduced risk of cancer and delayed progression of malignant disease. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-mTOR signaling is believed to partially contribute to these anticancer effects, although the mechanism is unclear. In this study, we revealed the mechanism underlying the effects of aspirin on AMPK-mTOR signaling, and described a mechanism-based rationale for the use of aspirin in cancer therapy. We found that aspirin inhibited mTORC1 signaling through AMPK-dependent and -independent manners. Aspirin inhibited the AMPK-TSC pathway, thus resulting in the suppression of mTORC1 activity. In parallel, it directly disrupted the mTOR-raptor interaction. Additionally, the combination of aspirin and sorafenib showed synergetic effects via inhibiting mTORC1 signaling and the PI3K/AKT, MAPK/ERK pathways. Aspirin and sorafenib showed synergetic anticancer efficacy in the SMMC-7721 model. Our study provides mechanistic insights and a mechanism-based rationale for the roles of aspirin in cancer treatment.

Aspirin Inhibits Cancer Metastasis and Angiogenesis via Targeting Heparanase.

Purpose: Recent epidemiological and clinical studies have suggested the benefit of aspirin for patients with cancer, which inspired increasing efforts to demonstrate the anticancer ability of aspirin and reveal the molecular mechanisms behind. Nevertheless, the anticancer activity and related mechanisms of aspirin remain largely unknown. This study aimed to confirm this observation, and more importantly, to investigate the potential target contributed to the anticancer of aspirin.Experimental Design: A homogeneous time-resolved fluorescence (HTRF) assay was used to examine the impact of aspirin on heparanase. Streptavidin pull-down, surface plasmon resonance (SPR) assay, and molecular docking were performed to identify heparanase as an aspirin-binding protein. Transwell, rat aortic rings, and chicken chorioallantoic membrane model were used to evaluate the antimetastasis and anti-angiogenesis effects of aspirin, and these phenotypes were tested in a B16F10 metastatic model, MDA-MB-231 metastatic model, and MDA-MB-435 xenograft model.Results: This study identified heparanase, an oncogenic extracellular matrix enzyme involved in cancer metastasis and angiogenesis, as a potential target of aspirin. We had discovered that aspirin directly binds to Glu225 region of heparanase and inhibits the enzymatic activity. Aspirin impeded tumor metastasis, angiogenesis, and growth in heparanase-dependent manner.Conclusions: In summary, this study has illustrated heparanase as a target of aspirin for the first time. It provides insights for a better understanding of the mechanisms of aspirin in anticancer effects, and offers a direction for the development of small-molecule inhibitors of heparanase. Clin Cancer Res; 23(20); 6267-78. ©2017 AACR.