Enhanced BCAT1 activity and BCAA metabolism promotes RhoC activity in cancer progression.

Increased expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) has been associated with aggressive phenotypes of different cancers. Here we identify a gain of function of BCAT1 glutamic acid to alanine mutation at codon 61 (BCAT1E61A) enriched around 2.8% in clinical gastric cancer samples. We found that BCAT1E61A confers higher enzymatic activity to boost branched-chain amino acid (BCAA) catabolism, accelerate cell growth and motility and contribute to tumor development. BCAT1 directly interacts with RhoC, leading to elevation of RhoC activity. Notably, the BCAA-derived metabolite, branched-chain α-keto acid directly binds to the small GTPase protein RhoC and promotes its activity. BCAT1 knockout-suppressed cell motility could be rescued by expressing BCAT1E61A or adding branched-chain α-keto acid. We also identified that candesartan acts as an inhibitor of BCAT1E61A, thus repressing RhoC activity and cancer cell motility in vitro and preventing peritoneal metastasis in vivo. Our study reveals a link between BCAA metabolism and cell motility and proliferation through regulating RhoC activation, with potential therapeutic implications for cancers.

Targeting metabolic vulnerability in mitochondria conquers MEK inhibitor resistance in KRAS-mutant lung cancer.

MEK is a canonical effector of mutant KRAS; however, MEK inhibitors fail to yield satisfactory clinical outcomes in KRAS-mutant cancers. Here, we identified mitochondrial oxidative phosphorylation (OXPHOS) induction as a profound metabolic alteration to confer KRAS-mutant non-small cell lung cancer (NSCLC) resistance to the clinical MEK inhibitor trametinib. Metabolic flux analysis demonstrated that pyruvate metabolism and fatty acid oxidation were markedly enhanced and coordinately powered the OXPHOS system in resistant cells after trametinib treatment, satisfying their energy demand and protecting them from apoptosis. As molecular events in this process, the pyruvate dehydrogenase complex (PDHc) and carnitine palmitoyl transferase IA (CPTIA), two rate-limiting enzymes that control the metabolic flux of pyruvate and palmitic acid to mitochondrial respiration were activated through phosphorylation and transcriptional regulation. Importantly, the co-administration of trametinib and IACS-010759, a clinical mitochondrial complex I inhibitor that blocks OXPHOS, significantly impeded tumor growth and prolonged mouse survival. Overall, our findings reveal that MEK inhibitor therapy creates a metabolic vulnerability in the mitochondria and further develop an effective combinatorial strategy to circumvent MEK inhibitors resistance in KRAS-driven NSCLC.

BCL6 is regulated by the MAPK/ELK1 axis and promotes KRAS-driven lung cancer.

Mutational activation of KRAS is a common oncogenic event in lung cancer, yet effective therapies are still lacking. Here, we identify B cell lymphoma 6 (BCL6) as a lynchpin in KRAS-driven lung cancer. BCL6 expression was increased upon KRAS activation in lung tumor tissue in mice and was positively correlated with the expression of KRAS-GTP, the active form of KRAS, in various human cancer cell lines. Moreover, BCL6 was highly expressed in human KRAS-mutant lung adenocarcinomas and was associated with poor patient survival. Mechanistically, the MAPK/ERK/ELK1 signaling axis downstream of mutant KRAS directly regulated BCL6 expression. BCL6 maintained the global expression of prereplication complex components; therefore, BCL6 inhibition induced stalling of the replication fork, leading to DNA damage and growth arrest in KRAS-mutant lung cancer cells. Importantly, BCL6-specific knockout in lungs significantly reduced the tumor burden and mortality in the LSL-KrasG12D/+ lung cancer mouse model. Likewise, pharmacological inhibition of BCL6 significantly impeded the growth of KRAS-mutant lung cancer cells both in vitro and in vivo. In summary, our findings reveal a crucial role of BCL6 in promoting KRAS-addicted lung cancer and suggest BCL6 as a therapeutic target for the treatment of this intractable disease.

Erk and MAPK signaling is essential for intestinal development through Wnt pathway modulation.

Homeostasis of intestinal stem cells (ISCs) is maintained by the orchestration of niche factors and intrinsic signaling networks. Here, we have found that deletion of Erk1 and Erk2 (Erk1/2) in intestinal epithelial cells at embryonic stages resulted in an unexpected increase in cell proliferation and migration, expansion of ISCs, and formation of polyp-like structures, leading to postnatal death. Deficiency of epithelial Erk1/2 results in defects in secretory cell differentiation as well as impaired mesenchymal cell proliferation and maturation. Deletion of Erk1/2 strongly activated Wnt signaling through both cell-autonomous and non-autonomous mechanisms. In epithelial cells, Erk1/2 depletion resulted in loss of feedback regulation, leading to Ras/Raf cascade activation that transactivated Akt activity to stimulate the mTor and Wnt/ß-catenin pathways. Moreover, Erk1/2 deficiency reduced the levels of Indian hedgehog and the expression of downstream pathway components, including mesenchymal Bmp4 - a Wnt suppressor in intestines. Inhibition of mTor signaling by rapamycin partially rescued Erk1/2 depletion-induced intestinal defects and significantly prolonged the lifespan of mutant mice. These data demonstrate that Erk/Mapk signaling functions as a key modulator of Wnt signaling through coordination of epithelial-mesenchymal interactions during intestinal development.

Blocking STAT3 by pyrvinium pamoate causes metabolic lethality in KRAS-mutant lung cancer.

Signal transducer and activator of transcription 3 (STAT3) exerts a profound role in regulating mitochondrial function and cellular metabolism. Mitochondrial STAT3 supports RAS-dependent malignant transformation and tumor growth. However, whether pharmacological blockade of STAT3 leads to metabolic lethality in KRAS-mutant lung cancer remains unclear. Pyrvinium pamoate, a clinical antihelminthic drug, preferentially inhibited the growth of KRAS-mutant lung cancer cells in vitro and in vivo. Mechanistic study revealed that pyrvinium dose-dependently suppressed STAT3 phosphorylation at tyrosine 705 and serine 727. Overexpression mitochondrial STAT3 prominently weakened the therapeutic efficacy of pyrvinium. As a result of targeting STAT3, pyrvinium selectively triggered reactive oxygen species release, depolarized mitochondrial membrane potential and suppressed aerobic glycolysis in KRAS-mutant lung cancer cells. Importantly, the cytotoxic effects of pyrvinium could be significantly augmented by glucose deprivation both in vitro and in a patient-derived lung cancer xenograft mouse model in vivo. The combined efficacy significantly correlated with intratumoural STAT3 suppression. Our findings reveal that KRAS-mutant lung cancer cells are vulnerable to STAT3 inhibition exerted by pyrvinium, providing a promising direction for developing therapies targeting STAT3 and metabolic synthetic lethality for the treatment of KRAS-mutant lung cancer.

Suppression of the SLC7A11/glutathione axis causes synthetic lethality in KRAS-mutant lung adenocarcinoma.

Oncogenic KRAS is a major driver in lung adenocarcinoma (LUAD) that has yet to be therapeutically conquered. Here we report that the SLC7A11/glutathione axis displays metabolic synthetic lethality with oncogenic KRAS. Through metabolomics approaches, we found that mutationally activated KRAS strikingly increased intracellular cystine levels and glutathione biosynthesis. SLC7A11, a cystine/glutamate antiporter conferring specificity for cystine uptake, was overexpressed in patients with KRAS-mutant LUAD and showed positive association with tumor progression. Furthermore, SLC7A11 inhibition by either genetic depletion or pharmacological inhibition with sulfasalazine resulted in selective killing across a panel of KRAS-mutant cancer cells in vitro and tumor growth inhibition in vivo, suggesting the functionality and specificity of SLC7A11 as a therapeutic target. Importantly, we further identified a potent SLC7A11 inhibitor, HG106, that markedly decreased cystine uptake and intracellular glutathione biosynthesis. Furthermore, HG106 exhibited selective cytotoxicity toward KRAS-mutant cells by increasing oxidative stress- and ER stress-mediated cell apoptosis. Of note, treatment of KRAS-mutant LUAD with HG106 in several preclinical lung cancer mouse models led to marked tumor suppression and prolonged survival. Overall, our findings reveal that KRAS-mutant LUAD cells are vulnerable to SLC7A11 inhibition, offering potential therapeutic approaches for this currently incurable disease.

Experimental study and mechanism analysis on the effect of substrate wettability on graphene sheets distribution morphology within uniform printing droplets.

Uniform graphene films and micro-patterns are the cornerstones of graphene-based printed electronics. However, disk-like reduced graphene oxide (RGO) sheets trend to concentrate at the edge of the drop because of the famous coffee-ring effect, resulting in non-uniform patterns. To understand the physics of coffee-ring formation for RGO droplets on hydrophilic substrates, we propose a mechanical model to elucidate the influence and its mechanism of substrate wettability on the solute migration behavior and solute distribution morphology of RGO droplets. Stronger coffee-ring morphology and a slower velocity transition on the PMMA can be observed as compared to that on the glass slides. An explanation based on the mechanical model is provided as the large contact angle on the PMMA leads to a small hindrance force and finally results in more significant coffee-ring morphology. Remarkably, we have revealed one underlying mechanism by which the hydrophilic substrate with better wettability will form more uniform patterns. This study can provide fundamental insights into the relationship between substrate wettability and RGO sheets distribution morphology. It might contribute to morphology regulation of RGO droplets in the DOD printing of graphene films and micro-patterns.

Autophagy inhibition enabled efficient photothermal therapy at a mild temperature.

The heterogeneously-distributed hyperthermia in nanomaterial-mediated photothermal therapy commonly results in incomplete tumor eradication and serious damage of health tissue. Here, we found autophagy was activated in cancer cells underwent photothermal therapy and the inhibition of autophagy significantly enhanced the efficacy of photothermal killing of cancer cells. A formulation of chloroquine-loaded polydopamine nanoparticles was developed for sensitized photothermal cancer therapy, and the in vitro and in vivo study demonstrated that inhibition of autophagy remarkably augmented the efficacy of photothermal therapy, leading to efficient tumor suppression at a mild temperature. The regulation of autophagy provides a new route to increase the efficacy of photothermal cancer therapy.

REGγ accelerates melanoma formation by regulating Wnt/ß-catenin signalling pathway.

It has been reported that the proteasome activator REGγ is associated with multiple oncogenic pathways in human cancers. However, the role of REGγ in the development of melanoma and the underlying mechanisms remain unclear. In this study, we attempted to investigate the effects of REGγ on human melanoma cell proliferation in vitro and in vivo. We demonstrated that knockdown of REGγ inhibited melanoma cell growth and arrested melanoma cell at G1 phase. Furthermore, depletion of REGγ also inhibited the xenograft growth of human melanoma. Mechanistically, REGγ activates Wnt/ß-catenin signal pathway by degrading GSK-3ß in melanoma cell lines and mouse models. Transient knockdown of ß-catenin effectively blocked cell proliferation in REGγ wild-type melanoma cells. In human melanoma samples, REGγ was overexpressed and positively correlated with ß-catenin levels. This study demonstrates that REGγ is a central molecule in the development of melanoma by regulating Wnt/ß-catenin pathway. This suggests that targeting REGγ could be an alternative therapeutic approach for melanoma.

FOXD3 is a tumor suppressor of colon cancer by inhibiting EGFR-Ras-Raf-MEK-ERK signal pathway.

Forkhead box D3 (FOXD3), as a transcriptional repressor, is well known to be involved in the regulation of development. Although FoxD3 is associated with several cancers, its role in colon cancer and the underlying mechanism are still unclear. Here, we first showed that FOXD3 knockdown dramatically increased the proliferation of human colon cancer cells, enhanced cell invasive ability and inhibited cell apoptosis. In vivo xenograft studies confirmed that the FOXD3-knockdown cells were more tumorigenic than the controls. Silencing FOXD3 markedly activated EGFR/Ras/Raf/MEK/ERK pathway in human colon cancer cells. In addition, blocking EGFR effectively decreased the activity of MAPK induced by FOXD3 knockdown. In human cancer tissue, the expression of FOXD3 was reduced, however, the EGFR/Ras/Raf/MEK/ERK pathway was activated. Our study indicates that FOXD3 may play a protective role in human colon formation by regulating EGFR/Ras/Raf/MEK/ERK signal pathway. It is proposed that FOXD3 may have potential as a new therapeutic target in human colon cancer treatment.

Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK.

No effective targeted therapies exist for cancers with somatic KRAS mutations. Here we develop a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results show that dual inhibition of polo-like kinase 1 and RhoA/Rho kinase (ROCK) leads to the synergistic effects in KRAS-mutant cancers. Microarray analysis reveals that this combinatory inhibition significantly increases transcription and activity of cyclin-dependent kinase inhibitor p21(WAF1/CIP1), leading to specific G2/M phase blockade in KRAS-mutant cells. Overexpression of p21(WAF1/CIP1), either by cDNA transfection or clinical drugs, preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and p21(WAF1/CIP1). Co-administration of BI-2536 and fasudil either in the LSL-KRAS(G12D) mouse model or in a patient tumour explant mouse model of KRAS-mutant lung cancer suppresses tumour growth and significantly prolongs mouse survival, suggesting a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers.

CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse.

The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies.