Bone transport induces the release of factors with multi-tissue regenerative potential for diabetic wound healing in rats and patients.

Tibial cortex transverse distraction is a surgical method for treating severe diabetic foot ulcers (DFUs), but the underlying mechanism is unclear. We show that antioxidant proteins and small extracellular vesicles (sEVs) with multiple-tissue regenerative potential are released during bone transport (BT) in humans and rats. These vesicles accumulate in diabetic wounds and are enriched with microRNAs (miRNAs) (e.g., miR-494-3p) that have high regenerative activities that improve the circulation of ischemic lower limbs while also promoting neovascularization, fibroblast migration, and nerve fiber regeneration. Deletion of miR-494-3p in rats reduces the beneficial effects of BT on diabetic wounds, while hydrogels containing miR-494-3p and reduced glutathione (GSH) effectively repair them. Importantly, the ginsenoside Rg1 can upregulate miR-494-3p, and a randomized controlled trial verifies that the regimen of oral Rg1 and GSH accelerates wound healing in refractory DFU patients. These findings identify potential functional factors for tissue regeneration and suggest a potential therapy for DFUs.

Mechanically stimulated osteocytes maintain tumor dormancy in bone metastasis of non-small cell lung cancer by releasing small extracellular vesicles.

Although preclinical and clinical studies have shown that exercise can inhibit bone metastasis progression, the mechanism remains poorly understood. Here, we found that non-small cell lung cancer (NSCLC) cells adjacent to bone tissue had a much lower proliferative capacity than the surrounding tumor cells in patients and mice. Subsequently, it was demonstrated that osteocytes, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing small extracellular vesicles with tumor suppressor micro-RNAs, such as miR-99b-3p. Furthermore, we evaluated the effects of mechanical loading and treadmill exercise on the bone metastasis progression of NSCLC in mice. As expected, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Notably, bone metastasis progression of NSCLC was inhibited by moderate exercise, and combinations with zoledronic acid had additive effects. Moreover, exercise preconditioning effectively suppressed bone metastasis progression. This study significantly advances the understanding of the mechanism underlying exercise-afforded protection against bone metastasis progression.

E74­like ETS transcription factor 5 facilitates cell proliferation through regulating the expression of adenomatous polyposis coli 2 in non­small cell lung cancer.

E74­like ETS transcription factor 5 (ELF5) is known to regulate the specification and differentiation of epithelial cells in the embryonic lung. However, the pathological function of ELF5 in lung cancer has yet to be fully elucidated. In the present study, the expression of ELF5 was found to be significantly higher in lung adenocarcinoma compared with that in corresponding adjacent normal tissues. Subsequently, cell and animal experiments were performed to investigate the role of ELF5 in lung adenocarcinoma cells. The results indicated that the overexpression of ELF5 increased the proliferation of lung adenocarcinoma cells, whereas, by contrast, a reduction in the expression of ELF5 led to a decrease in their proliferation. Mechanistically, the hypothesis is advanced that ELF5 can promote lung cancer cell proliferation through inhibiting adenomatous polyposis coli 2 and increasing the expression of cyclin D1, which is a critical downstream target of the Wnt pathway. Taken together, these findings support the notion that ELF5 exerts an essential role in the proliferation of lung adenocarcinoma cells and may be a therapeutic target for the treatment of lung adenocarcinoma.

Damaged brain accelerates bone healing by releasing small extracellular vesicles that target osteoprogenitors.

Clinical evidence has established that concomitant traumatic brain injury (TBI) accelerates bone healing, but the underlying mechanism is unclear. This study shows that after TBI, injured neurons, mainly those in the hippocampus, release osteogenic microRNA (miRNA)-enriched small extracellular vesicles (sEVs), which targeted osteoprogenitors in bone to stimulate bone formation. We show that miR-328a-3p and miR-150-5p, enriched in the sEVs after TBI, promote osteogenesis by directly targeting the 3'UTR of FOXO4 or CBL, respectively, and hydrogel carrying miR-328a-3p-containing sEVs efficiently repaires bone defects in rats. Importantly, increased fibronectin expression on sEVs surface contributes to targeting of osteoprogenitors in bone by TBI sEVs, thereby implying that modification of the sEVs surface fibronectin could be used in bone-targeted drug delivery. Together, our work unveils a role of central regulation in bone formation and a clear link between injured neurons and osteogenitors, both in animals and clinical settings.

Osteocytes regulate neutrophil development through IL-19: a potent cytokine for neutropenia treatment.

Osteocytes are the most abundant (90% to 95%) cells in bone and have emerged as an important regulator of hematopoiesis, but their role in neutrophil development and the underlying mechanisms remain unclear. Interleukin 19 (IL-19) produced predominantly by osteocytes stimulated granulopoiesis and neutrophil formation, which stimulated IL-19 receptor (IL-20Rß)/Stat3 signaling in neutrophil progenitors to promote their expansion and neutrophil formation. Mice with constitutive activation of mechanistic target of rapamycin complex (mTORC1) signaling in osteocytes (Dmp1-Cre) exhibited a dramatic increase in IL-19 production and promyelocyte/myelocytic expansion, whereas mTORC1 inactivation in osteocytes reduced IL-19 production and neutrophil numbers in mice. We showed that IL-19 administration stimulated neutrophil development, whereas neutralizing endogenous IL-19 or depletion of its receptor inhibited the process. Importantly, low-dose IL-19 reversed chemotherapy, irradiation, or chloramphenicol-induced neutropenia in mice more efficiently than granulocyte colony-stimulating factor. This evidence indicated that IL-19 was an essential regulator of neutrophil development and a potent cytokine for neutropenia treatment.

ETS2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting JUNB transcription.

Epithelial-to-mesenchymal transition (EMT) plays an important role in the progression of renal tubulointerstitial fibrosis, a common mechanism leading to end-stage renal failure. V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2), a transcription factor, exhibits diverse roles in pathogenesis; however, its role in renal fibrosis is not yet fully understood. In this study, we detected the expression of ETS2 in an animal model of renal fibrosis and evaluated the potential role of ETS2 in tubular EMT induced by TGF-ß1. We found that ETS2 and profibrogenic factors, alpha-smooth muscle actin (α-SMA) and fibronectin (FN), were significantly increased in the unilateral ureteral obstruction (UUO)-induced renal fibrosis model in mice. In vitro, TGF-ß1 induced a high expression of ETS2 dependent on Smad3 and ERK signaling pathway in human proximal tubular epithelial cells (HK2). Knockdown of ETS2 abrogated TGF-ß1-mediated expression of profibrogenic factors vimentin, α-SMA, collagen I, and FN in HK2 cells. Mechanistically, ETS2 promoted JUNB expression in HK2 cells after TGF-ß1 stimulation. Furthermore, luciferase and Chromatin Immunoprecipitation (ChIP) assays revealed that the binding of ETS2 to three EBS motifs on the promoter of JUNB triggered its transcription. Notably, silencing JUNB reversed the ETS2-induced upregulation of the profibrogenic factors in HK2 cells after TGF-ß1 stimulation. These findings suggest that ETS2 mediates TGF-ß1-induced EMT in renal tubular cells through JUNB, a novel pathway for preventing renal fibrosis.

Exosome Release Is Regulated by mTORC1.

Exosomes are small membrane-bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA, and miRNA, and are involved in cell waste management and intercellular communication. In the present study, it is shown that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). It is found that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63-positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug-stimulated release is blocked by siRNA-mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin-treated cells reveals that inhibition of mTORC1 does not significantly alter its majority protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions.

Raptor directs Sertoli cell cytoskeletal organization and polarity in the mouse testis.

Sertoli cells (SCs) play a central role in testis development, and their normal number and functions are required for spermatogenesis. Although the canonical tuberous sclerosis complex-mammalian target of rapamycin complex 1(TSC-mTORC1) pathway is critical for testis development and spermatogenesis, the signaling mechanisms governing SC functions remain unclear. In this study, we generated two SC-specific mouse mutants using the Cre-LoxP system. Loss of Raptor (a key component of mTORC1) caused severe tubular degeneration in the neonatal testis and adult mice displayed azoospermia, while adult Rheb (an upstream activator for mTORC1) mutant mice had intact tubules and many sperm in their epididymides. Disruption of cytoskeletal organization, including actin, microtubules, and SC-intrinsic vimentin, was observed in Raptor but not Rheb mutant mice. We investigated the reasons for these different effects by whole-transcriptome sequencing, and found that expression of the tight junction adaptor protein cingulin was significantly reduced in Raptor mutant mice. The expression profile of cingulin was synchronous with the differentiation and cytoskeletal dynamics of SCs in control mice, but was disordered in Raptor mutant mice. Furthermore, activity of the small GTPase Rac1 was reduced and expression of the guanine exchange factor for Rac1, Asef, was decreased in Raptor but not Rheb mutant mice. Collectively, these findings establish novel functions of Raptor, independent of the canonical Rheb/mTORC1 pathway, in controlling cytoskeletal homeostasis and cell polarity in SCs, by affecting cingulin expression and Rac1 activity.

Bone and plasma citrate is reduced in osteoporosis.

High concentration of citrate exists in bone of humans and all osteo-vertebrates, and citrate incorporation imparts important biomechanical and other functional properties to bone. However, which cells are responsible for citrate production in bone remains unclear and whether the citrate component changes with bone loss during osteoporosis is also not known. Here, we show that the citrate content is markedly reduced in the bone of mice or rats with age-related, ovariectomy-induced or retinoic acid-induced bone loss. Plasmic citrate is also downregulated in osteoporotic animals. Importantly, the plasmic citrate level of aged osteoporotic males is significantly lower than that of young healthy males and positively correlates with human lumbar spine bone mineral density (BMD) and total hip BMD. Furthermore, citrate production increases with in vitro osteoblastic differentiation, accompanied by upregulation of proteins involved in citrate secretion, suggesting that osteoblasts are highly specialized cells that produce citrate in bone. Our findings establish a novel relationship between citrate content and bone loss-related diseases such as osteoporosis, suggesting a critical role of bone citrate in the maintenance of the citrate balance in the circulation. Serum citrate level may thus represent a novel marker for osteoporosis.

Loss of DEPTOR in renal tubules protects against cisplatin-induced acute kidney injury.

DEP domain containing mTOR-interacting protein (DEPTOR) was originally identified as an in vivo dual inhibitor of mechanistic target of rapamycin (mTOR). It was recently reported to be involved in renal physiology and pathology in vitro; however, its detailed roles and mechanisms in vivo are completely unknown. We observed that DEPTOR expression in the kidney was markedly increased on day 3 after cisplatin treatment, at which time cell apoptosis peaked, implicating DEPTOR in cisplatin-induced acute kidney injury (AKI). We then used the Cre-LoxP system to generate mutant mice in which the DEPTOR gene was specifically deleted in the proximal tubule cells. DEPTOR deficiency did not alter the renal histology or functions in the saline-treated group, indicating that DEPTOR is not essential for kidney function under physiological conditions. Interestingly, DEPTOR deletion extensively preserved the renal histology and maintained the kidney functions after cisplatin treatment, suggesting that the absence of DEPTOR ameliorates cisplatin-induced AKI. Mechanistically, DEPTOR modulated p38 MAPK signaling and TNFα production in vivo and in vitro, rather than mTOR signaling, thus moderating the inflammatory response and cell apoptosis induced by cisplatin. Collectively, our findings demonstrate the roles and mechanisms of DEPTOR in the regulation of the renal physiology and pathology, and demonstrate that the loss of DEPTOR in the proximal tubules protects against cisplatin-induced AKI.

Tyrosine kinase Fyn promotes osteoarthritis by activating the ß-catenin pathway.

OBJECTIVES: To investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice. METHODS: Cartilage samples from normal and aged mice were analysed with proteome-wide screening. Fyn expression was examined with immunofluorescence in human and age-dependent or experimental mouse OA cartilage samples. Experimental OA in Fyn-knockout mice was induced by destabilisation of the medial meniscus. Primary cultured mouse chondrocytes were treated with proinflammatory cytokine interleukin-1ß. The inhibitor of Src kinase family, AZD0530 (saracatinib), and inhibitor of Fyn, PP1, were used to treat experimental OA in mice. RESULTS: Fyn expression was markedly upregulated in human OA cartilage and in cartilage from aged mice and those with post-traumatic OA. Fyn accumulates in articular chondrocytes and interacts directly with and phosphorylates ß-catenin at Tyr142, which stabilises ß-catenin and promotes its nuclear translocation. The deletion of Fyn effectively delayed the development of post-traumatic and age-dependent OA in mice. Fyn inhibitors AZD0530 and PP1 significantly attenuated OA progression by blocking the ß-catenin pathway and reducing the levels of extracellular matrix catabolic enzymes in the articular cartilage. CONCLUSIONS: Fyn accumulates and activates ß-catenin signalling in chondrocytes, accelerating the degradation of the articular cartilage and OA development. Targeting Fyn is a novel and potentially therapeutic approach to the treatment of OA.

Loss of Rictor with aging in osteoblasts promotes age-related bone loss.

Osteoblast dysfunction is a major cause of age-related bone loss, but the mechanisms underlying changes in osteoblast function with aging are poorly understood. This study demonstrates that osteoblasts in aged mice exhibit markedly impaired adhesion to the bone formation surface and reduced mineralization in vivo and in vitro. Rictor, a specific component of the mechanistic target of rapamycin complex 2 (mTORC2) that controls cytoskeletal organization and cell survival, is downregulated with aging in osteoblasts. Mechanistically, we found that an increased level of reactive oxygen species with aging stimulates the expression of miR-218, which directly targets Rictor and reduces osteoblast bone surface adhesion and survival, resulting in a decreased number of functional osteoblasts and accelerated bone loss in aged mice. Our findings reveal a novel functional pathway important for age-related bone loss and support for miR-218 and Rictor as potential targets for therapeutic intervention for age-related osteoporosis treatment.

[Application of mesoesophagus suspension technique in upper mediastinal lymph node dissection during thoracoscopic esophagectomy].

OBJECTIVE: To explore the application of mesoesophagus suspension technique to improve the upper mediastinal lymph node dissection during thoracoscopic esophagectomy in the treatment of esophageal cancer. METHODS: Clinical data of 164 thoracic esophageal cancer patients who underwent combined thoracoscopic and laparoscopic esophagectomy with two-field lymph node dissection in the Union Hospital of Fujian Medical University between October 2012 and June 2015 were retrospectively analyzed. Among 164 patients, 80 cases underwent upper mediastinal lymph node dissection by traditional method (traditional group), and the remaining 84 cases underwent upper mediastinal lymph node dissection by mesoesophagus suspension technique (suspension group). The operation time, estimated blood loss, number of excised lymph nodes and postoperative complications were compared between the two groups. RESULTS: There were no significant differences in gender, age, location of tumor and pathology stage between the two groups. The operation time in the two groups was similar. The suspension group had significantly less thoracic blood loss than traditional group [(85±5) ml vs.(140±7) ml, P=0.000]. The number of dissected lymph nodes of bilateral recurrent laryngeal nerve was more in suspension group [median (interquartile range): left: 3 (2 to 4) vs. 2 (1 to 3), P=0.013; right: 3(2 to 6) vs. 2(1 to 3), P=0.007]. There was no significant difference in metastatic rate of lymph node in different sites between the two groups. The highest metastatic rate of suspension and traditional group was found at paracardia lymph nodes[22.6%(19/84) and 22.5%(18/80)], the next was at right laryngeal nerve lymph nodes [17.9%(15/84) and 15.0%(12/80)] and left laryngeal nerve lymph nodes [16.7%(14/84) and 12.5%(10/80)]. There were no significant differences with regard to the incidence of major postoperative complications between two groups, including respiratory complication, anastomotic leakage, vocal cord palsy. CONCLUSIONS: Upper mediastinal bilateral recurrent laryngeal nerve lymph node is the predilection site of lymphatic metastasis of thoracic esophageal cancer. Application of mesoesophagus suspension technique in thoracoscopic esophagectomy can improve the clearance quality of bilateral recurrent laryngeal nerve lymph nodes.

MicroRNA-218 inhibits EMT, migration and invasion by targeting SFMBT1 and DCUN1D1 in cervical cancer.

Repeated infection with high-risk HPV is a major cause for the development and metastasis of human cervical cancer, even though the mechanism of the metastasis is still not completely understood. Here, we reported that miR-218 (microRNA-218) was downregulated in cervical cancer tissues, especially in metastatic cancer tissues. We found that miR-218 expression was associated with clinicopathological characteristics of patients with cervical cancer. MiR-218 overexpression inhibited Epithelial-Mesenchymal Transition (EMT), migration and invasiveness of cervical cancer cells in vitro. Moreover, miR-218 repressed the expression of SFMFBT1 (Scm-like with four MBT domains 1) and DCUN1D1 (defective in cullin neddylation 1, domain containing 1) by direct binding to the 3'UTRs of the mRNAs. The overexpression of SFMBT1 induced EMT and increased the migration and invasiveness of cervical cancer cells, while the overexpression of DCUN1D1 increased the migration and invasiveness of these cells, but did not induce EMT. An inverse correlation was observed between the expression of miR-218 and DCUN1D1 protein in cervical cancer tissues. Importantly, HPV16 E6 downregulated the expression of miR-218 in cervical cancer, while miR-218 rescued the promotion effect of HPV16 E6 on the expression of SFMBT1 and DCUN1D1. Taken together, our results revealed that HPV16 E6 promoted EMT and invasion in cervical cancer via the repression of miR-218, while miR-218 inhibited EMT and invasion in cervical cancer by targeting SFMBT1 and DCUN1D1.

mTORC2 promotes cell survival through c-Myc-dependent up-regulation of E2F1.

Previous studies have reported that mTORC2 promotes cell survival through phosphorylating AKT and enhancing its activity. We reveal another mechanism by which mTORC2 controls apoptosis. Inactivation of mTORC2 promotes binding of CIP2A to PP2A, leading to reduced PP2A activity toward c-Myc serine 62 and, consequently, enhancement of c-Myc phosphorylation and expression. Increased c-Myc activity induces transcription of pri-miR-9-2/miR-9-3p, in turn inhibiting expression of E2F1, a transcriptional factor critical for cancer cell survival and tumor progression, resulting in enhanced apoptosis. In vivo experiments using B cell-specific mTORC2 (rapamycin-insensitive companion of mTOR) deletion mice and a xenograft tumor model confirmed that inactivation of mTORC2 causes up-regulation of c-Myc and miR-9-3p, down-regulation of E2F1, and consequent reduction in cell survival. Conversely, Antagomir-9-3p reversed mTORC1/2 inhibitor-potentiated E2F1 suppression and resultant apoptosis in xenograft tumors. Our in vitro and in vivo findings collectively demonstrate that mTORC2 promotes cell survival by stimulating E2F1 expression through a c-Myc- and miR-9-3p-dependent mechanism.

Serum microRNA-218 is a potential biomarker for esophageal cancer.

BACKGROUND: Several studies demonstrated that microRNAs are stably detectable in plasma/serum and are potential biomarkers for some diseases. The expression of microRNA-218 (miR-218) is downregulated in esophageal cancer as reported in previous research. OBJECTIVE: The purpose of this study is to investigate whether miR-218 can be served as a serum biomarker for esophageal cancer. METHODS: We tested the expression level of miR-218 in serum of 106 patients with esophageal cancer and 60 healthy volunteers by RT-PCR and analyzed the relationship between serum miR-218 expression and the clinical characteristics. RESULTS: The serum expression of miR-218 was significantly lower in patients with esophageal cancer than that in healthy individuals. The value of the area under the receiver-operating characteristic (ROC) curve (AUC) was 0.833. Furthermore, the ROC curves to detect early esophageal cancer with Tis-T1 or Stage 0-I showed AUCs of 0.825 and 0.829, respectively. In the esophageal cancer group, the serum expression of miR-218 was found to be lower in esophageal cancer patients with poorer differentiation, later stage, and lymph node metastasis, highlighting that low serum expression of miR-218 may be related to tumor development and progression in esophageal cancer. CONCLUSIONS: The serum expression of miR-218 is downregulated in esophageal cancer patients and is correlated with tumor differentiation, stage, and lymph node metastasis. Serum miR-218 may be a potential biomarker for early detection and clinical evaluation in patient with esophageal cancer.

miR-483-5p promotes invasion and metastasis of lung adenocarcinoma by targeting RhoGDI1 and ALCAM.

The nodal regulatory properties of microRNAs (miRNA) in metastatic cancer may offer new targets for therapeutic control. Here, we report that upregulation of miR-483-5p is correlated with the progression of human lung adenocarcinoma. miR-483-5p promotes the epithelial-mesenchymal transition (EMT) accompanied by invasive and metastatic properties of lung adenocarcinoma. Mechanistically, miR-483-5p is activated by the WNT/ß-catenin signaling pathway and exerts its prometastatic function by directly targeting the Rho GDP dissociation inhibitor alpha (RhoGDI1) and activated leukocyte cell adhesion molecule (ALCAM), two putative metastasis suppressors. Furthermore, we found that downregulation of RhoGDI1 enhances expression of Snail, thereby promoting EMT. Importantly, miR-483-5p levels are positively correlated with ß-catenin expression, but are negatively correlated with the levels of RhoGDI1 and ALCAM in human lung adenocarcinoma. Our findings reveal that miR-483-5p is a critical ß-catenin-activated prometastatic miRNA and a negative regulator of the metastasis suppressors RhoGDI1 and ALCAM.

Activation of mTORC1 in collecting ducts causes hyperkalemia.

Mutation of TSC (encoding tuberous sclerosis complex protein) and activation of mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of several renal diseases, such as diabetic nephropathy and polycystic kidney disease. However, the role of mTOR in renal potassium excretion and hyperkalemia is not known. We showed that mice with collecting-duct (CD)-specific ablation of TSC1 (CDTsc1KO) had greater mTOR complex 1 (mTORC1) activation in the CD and demonstrated features of pseudohypoaldosteronism, including hyperkalemia, hyperaldosteronism, and metabolic acidosis. mTORC1 activation caused endoplasmic reticulum stress, columnar cell lesions, and dedifferentiation of CD cells with loss of aquaporin-2 and epithelial-mesenchymal transition-like phenotypes. Of note, mTORC1 activation also reduced the expression of serum- and glucocorticoid-inducible kinase 1, a crucial regulator of potassium homeostasis in the kidney, and decreased the expression and/or activity of epithelial sodium channel-α, renal outer medullary potassium channel, and Na(+), K(+)-ATPase in the CD, which probably contributed to the aldosterone resistance and hyperkalemia in these mice. Rapamycin restored these phenotypic changes. Overall, this study identifies a novel function of mTORC1 in regulating potassium homeostasis and demonstrates that loss of TSC1 and activation of mTORC1 results in dedifferentiation and dysfunction of the CD and causes hyperkalemia. The CDTsc1KO mice provide a novel model for hyperkalemia induced exclusively by dysfunction of the CD.

miR-124 represses ROCK1 expression to promote neurite elongation through activation of the PI3K/Akt signal pathway.

Recent studies have demonstrated an important role for miR-124, the most abundant and well-conserved brain-specific microRNA(miRNA), in promoting neurite outgrowth and elongation during neuronal differentiation. This miRNA's target genes and the mechanisms that execute this role remain unclear. In this study, we identified ROCK1, a small GTPase Rho kinase, as a direct target of miR-124 for regulating neurite elongation. miR-124 significantly inhibited ROCK1 expression in M17 cells. Inhibiting ROCK1 promoted neurite elongation, and the overexpression of ROCK1 strongly repressed the neurite elongation-enhancing effect of miR-124 in M17 cells. We determined that Akt functions as a novel ROCK1 downstream effector in regulating neurite outgrowth and elongation.

mTORC1 is a target of nordihydroguaiaretic acid to prevent breast tumor growth in vitro and in vivo.

Nordihydroguaiaretic acid (NDGA) is a natural phenolic compound isolated from the creosote bush Larrea divaricata, which has anti-tumor activities both in vitro and in vivo. Its analogs are in clinical development for use in refractory solid tumors. But the mechanisms underlying the anti-cancer effect of NDGA are not fully understood. In this study, we identified mammalian target of rapamycin complex 1 (mTORC1) as a target of NDGA both in cultured breast cancer cells and in xenograft models. NDGA effectively inhibited basal level of mTORC1 but not mTORC2 activity in breast cancer cell lines. NDGA also suppressed mTORC1 downstream signaling such as expression of cyclin D1, hypoxia-inducible factor-α and VEGF, and prevented proliferation in breast cancer cells. Although NDGA stimulated AMP-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2) signaling, which negatively regulates mTORC1, AMPK and TSC2 deletion could not diminish the inhibition of mTORC1 by NDGA. Subsequent studies revealed that NDGA may also direct target mTORC1 complex because NDGA suppressed amino acids- and insulin-stimulated mTORC1 and acted like rapamycin to disrupt mTOR-Raptor interaction. Most importantly, NDGA repressed breast tumor growth and targeted mTORC1 and its downstream signaling in xenograft models. Together our data provide a novel mechanism for NDGA activity which could help explain its anti-cancer activity. Disruption of mTOR-Raptor complex and activation of AMPK/TSC signaling may contribute to inhibitory effects of NDGA against mTORC1. Our data also raise the possibility that NDGA, as an mTORC1 inhibitor, may have a broad spectrum of action on breast cancers.