SLC7A2 deficiency promotes hepatocellular carcinoma progression by enhancing recruitment of myeloid-derived suppressors cells

The main reason for poor prognosis in hepatocellular carcinoma (HCC) patients is high metastasis and recurrence. Cancer progression depends on a tumor-supportive microenvironment. Therefore, illustrating the mechanisms of tumor immunity in underlying HCC metastasis is essential. Here, we report a novel role of solute carrier family 7 member 2 (SLC7A2), a member of the solute carrier family, in HCC metastasis. The reduction of SLC7A2 was an independent and significant risk factor for the survival of HCC patients. Upregulation of SLC7A2 decreased HCC invasion and metastasis, whereas downregulation of SLC7A2 promoted HCC invasion and metastasis. We further found that deficient SLC7A2 medicated the upregulation of CXCL1 through PI3K/Akt/NF-kκB pathway to recruit myeloid-derived suppressor cells (MDSCs), exerting tumor immunosuppressive effect. Moreover, we found that G9a-mediated di-methylation of H3K9 (H3K9me2) silenced the expression of SLC7A2 to suppress HCC metastasis and immune escape. In conclusion, G9a-mediated silencing of SLC7A2 exerts unexpected functions in cancer metastasis by fostering a tumor-supportive microenvironment through CXCL1 secretion and MDSCs recruitment. Thus, SLC7A2 may provide new mechanistic insight into the cancer-promoting property of MDSCs.Subject terms: Cancer microenvironment, Cancer immunotherapy, Metastasis     

HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination-mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.     

Pin1 interacts with c-Myb in a phosphorylation-dependent manner and regulates its transactivation activity

Activity and stability of the proto-oncogene c-Myb are regulated by post-translational modifications, though the molecular mechanisms underlying such control are only partially understood. Here we describe the functional interaction of c-Myb with Pin1, an isomerase that binds to phosphorylated Ser/Thr-Pro motifs. We found that co-expression of c-Myb and Pin1 led to a net increase of c-Myb transactivation activity, both on reporter constructs as well as on an endogenous target gene. DNA-binding studies revealed that Pin1 did not increase the association of c-Myb with its response element in DNA. The increase of c-Myb transactivation activity was strictly dependent on the presence of an active catalytic center in Pin1. We provide evidence that c-Myb and Pin1 physically interacted, both upon ectopic expression of the proteins in HEK-293 cells as well as in the more physiological setting of HL60 cells, where c-Myb and Pin1 are resident proteins. By point mutating each individual Ser/Thr-Pro motif in c-Myb as well as by using deletion mutants we show that S528 in the EVES-motif was the docking site for Pin1. Mass spectrometry confirmed that S528 is phosphorylated in vivo. Finally, functional studies showed that mutation of S528 to alanine almost abolished the increase of transactivation activity by Pin1. This study reveals a new paradigm by which phosphorylation controls c-Myb function.     

Cortactin promotes cell motility by enhancing lamellipodial persistence

BACKGROUND: Lamellipodial protrusion, which is the first step in cell movement, is driven by actin assembly and requires activity of the Arp2/3 actin-nucleating complex. However, it is unclear how actin assembly is dynamically regulated to support effective cell migration. RESULTS: Cells deficient in cortactin have impaired cell migration and invasion. Kymography analyses of live-cell imaging studies demonstrate that cortactin-knockdown cells have a selective defect in the persistence of lamellipodial protrusions. The motility and protrusion defects are fully rescued by cortactin molecules, provided both the Arp2/3 complex and F-actin binding sites are intact. Consistent with this requirement for simultaneous contacts with Arp2/3 and F-actin, cortactin is recruited by Arp2/3 complex to lamellipodia and binds with a higher affinity to ATP/ADP-Pi-F-actin than to ADP-F-actin. In situ labeling of lamellipodia revealed that the relative levels of free barbed ends of actin filaments are reduced by over 30% in the cortactin-knockdown cells; however, there is no change in Arp2/3-complex localization to lamellipodia. Cortactin-knockdown cells also have a selective defect in the assembly of new adhesions in protrusions, as assessed by analysis of GFP-paxillin dynamics in living cells. CONCLUSIONS: Cortactin enhances lamellipodial persistence, at least in part through regulation of Arp2/3 complex. The presence of cortactin also enhances the rate of new adhesion formation in lamellipodia. In vivo, these functions may be important during directed cell motility.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

GSI promotes vincristine-induced apoptosis by enhancing multi-polar spindle formation

Gamma secretase inhibitors (GSI), cell-permeable small-molecule inhibitors of gamma secretase activity, had been originally developed for the treatment of Alzheimer disease. In recent years, it has been exploited in cancer research to inhibit Notch signaling that is aberrantly activated in various cancers. We previously found that GSI could synergize with anti-microtubule agent, vincristine (VCR) in a Notch-independent manner. Here, we delineate the underlying cell cycle-related mechanism using HeLa cells, which have strong mitotic checkpoints. GSI enhanced VCR-induced cell death, although GSI alone did not affect cell viability at all. GSI augmented VCR-induced mitotic arrest in a dose-dependent manner, which was preceded by apoptotic cell death, as shown by an increase in Annexin V-positive and caspase-positive cell population. Furthermore, GSI amplified multi-polar spindle formation triggered by VCR. Altogether, we show the evidence that GSI enhances VCR-induced apoptosis in HeLa cells via multi-polar mitotic spindle formation, independent of Notch signaling. These data suggest that one or more GS substrates, yet to be identified, in a post-GS processed form, may play a role in maintaining functional centrosomes/mitotic spindles. More significantly, the synergistic effect of GSI in combination with VCR could be exploited in clinical setting to improve the efficacy of VCR.     

Bag-1M inhibits the transactivation of the glucocorticoid receptor via recruitment of corepressors

The Bcl-2 associated athanogene 1M (Bag-1M) is known to repress the transactivation of the glucocorticoid receptor (GR). We report here that Bag-1M inhibits the action of GR via recruitment of corepressors, including nuclear receptor corepressor (NcoR) and silencing mediator for retinoic acid and thyroid hormone receptor (SMRT), and histone deacetylase (HDAC)3 to the genomic response element of a glucocorticoid-regulated human metallothionein IIa (hMTIIa) gene. A mutant GR lacking the interaction with BAG-1M fails to recruit the corepressors NcoR and SMRT. RNAi-mediated knock down of corepressors and the use of HDAC inhibitor relieved Bag-1M-induced repression on the transactivation of the GR. In addition, Bag-1M is not involved in the degradation of the receptor. These findings indicate a novel mechanism by which Bag-1M acts as a corepressor and downregulates the activity of the GR.

Structured summary

MINT-7216164: HDAC3 (uniprotkb:O15379) physically interacts (MI:0914) with Bag1 (uniprotkb:Q99933) by anti bait coimmunoprecipitation (MI:0006)MINT-7216183: NCOR (uniprotkb:O75376) physically interacts (MI:0914) with Bag1 (uniprotkb:Q99933) by anti bait coimmunoprecipitation (MI:0006)MINT-7216175: SMRT (uniprotkb:Q9Y618) physically interacts (MI:0914) with Bag1 (uniprotkb:Q99933) by anti bait coimmunoprecipitation (MI:0006)