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Cancer cells are defective in DNA repair, so they experience increased DNA strand breaks, genome instability, gene mutagenesis, and tumorigenicity; however, multiple classic DNA repair genes and pathways are strongly activated in malignant tumor cells to compensate for the DNA repair deficiency and gain an apoptosis resistance. The mechanisms underlying this phenomenon in cancer are unclear. We speculate that a key DNA repair gene or signaling pathway in cancer has not yet been recognized. Here, we show that the lipogenic liver X receptor (LXR)-sterol response element binding factor-1 (SREBF1) axis controls the transcription of a key DNA repair gene polynucleotide kinase/phosphatase (PNKP), thereby governing cancer cell DNA repair and apoptosis. Notably, the PNKP levels were significantly reduced in 95% of human pancreatic cancer (PC) patients, particularly deep reduction for sixfold in all of the advanced-stage PC cases. PNKP is also deficient in three other types of cancer that we examined. In addition, the expression of LXRs and SREBF1 was significantly reduced in the tumor tissues from human PC patients compared with the adjacent normal tissues. The newly identified LXR-SREBF1-PNKP signaling pathway is deficient in PC, and the defect in the pathway contributes to the DNA repair deficiency in the cancer. Strikingly, further diminution of the vulnerable LXR-SREBF1-PNKP signaling pathway using a small molecule triptonide, a new LXR antagonist identified in this investigation, at a concentration of 8 nM robustly activated tumor-suppressor p53 and readily elevated cancer cell DNA strand breaks over an apoptotic threshold, and selectively induced PC cell apoptosis, resulting in almost complete elimination of tumors in xenograft mice without obvious complications. Our findings provide new insight into DNA repair and apoptosis in cancer, and offer a new platform for developing novel anticancer therapeutics.

Background Triple-negative breast cancer (TNBC) is highly metastatic and lethal. Due to a lack of druggable targets for this disease, there are no effective therapies in the clinic. Methods We used TNBC cells and xenografted mice as models to explore triptonide-mediated inhibition of TNBC metastasis and tumor growth. Colony formation assay was used to quantify the tumorigenesis of TNBC cells. Wound-healing and cell trans-well assays were utilized to measure cell migration and invasion. Tube formation assay was applied to access tumor cell-mediated vasculogenic mimicry. Western blot, quantitative-PCR, immunofluorescence imaging, and immunohistochemical staining were used to measure the expression levels of various tumorigenic genes in TNBC cells. Results Here, we showed that triptonide, a small molecule from the traditional Chinese medicinal herb Tripterygium wilfordii Hook F, potently inhibited TNBC cell migration, invasion, and vasculogenic mimicry, and effectively suppressed TNBC tumor growth and lung metastasis in xenografted mice with no observable toxicity. Molecular mechanistic studies revealed that triptonide strongly triggered the degradation of master epithelial-mesenchymal transition (EMT)-inducing protein Twist1 through the lysosomal system and reduced Notch1 expression and NF-κB phosphorylation, which consequently diminished the expression of pro-metastatic and angiogenic genes N-cadherin, VE-cadherin, and vascular endothelial cell growth factor receptor 2 (VEGFR2). Conclusions Triptonide effectively suppressed TNBC cell tumorigenesis, vasculogenic mimicry, and strongly inhibited the metastasis of TNBC via degradation of Twist1 and Notch1 oncoproteins, downregulation of metastatic and angiogenic gene expression, and reduction of NF-κB signaling pathway. Our findings provide a new strategy for treating highly lethal TNBC and offer a potential new drug candidate for combatting this aggressive disease.

Transmembrane serine proteases have been implicated in the development and progression of solid and hematological cancers. Human airway trypsin‐like protease 4 (HAT‐L4) is a transmembrane serine protease expressed in epithelial cells and exocrine glands. In the skin, HAT‐L4 is important for normal epidermal barrier function. Here, we report an unexpected finding of ectopic HAT‐L4 expression in neutrophils and monocytes from acute myeloid leukemia (AML) patients. Such expression was not detected in bone marrow cells from normal individuals or patients with chronic myeloid leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. In AML patients who underwent chemotherapy, persistent HAT‐L4 expression in bone marrow cells was associated with minimal residual disease and poor prognostic outcomes. In culture, silencing HAT‐L4 expression in AML–derived THP‐1 cells by short hairpin RNAs inhibited matrix metalloproteinase‐2 activation and Matrigel invasion. In mouse xenograft models, inhibition of HAT‐L4 expression reduced the proliferation and growth of THP‐1 cell–derived tumors. Our results indicate that ectopic HAT‐L4 expression is a pathological mechanism in AML and that HAT‐L4 may be used as a cell surface marker for AML blast detection and targeting.

Lung cancer is a leading lethal disease with a 5-year survival rate of only 16%. Inadequate potent anti-cancer drugs appear to be a bottleneck in the treatment of lung cancer; hence, how to develop effective anti-lung cancer therapeutics is an urgent problem. In this study, we aim to explore a novel compound with potent anti-lung cancer effect and study its anti-cancer mechanisms. We found that triptonide at very low concentrations of 5–10 nM caused a marked suppression of cell proliferation and colony formation of lung cancer cells. More interestingly, triptonide also robustly inhibited the lung cancer cell formation of tumor spheres, and reduced the stemness and tumorigenicity of the sphere-forming cells. In vivo studies showed that administration of triptonide significantly inhibited the tumor growth with low toxicity. Molecular mechanistic studies revealed that triptonide significantly decreased expression of the Gli1 at both mRNA and protein levels by repressing Gli1 gene promoter activity. Additionally, triptonide reduced the levels of cancer stem cell key signaling protein sonic hedgehog (Shh), but increased the amount of Ptch1, a protein binding to SMO to diminish the Shh signal transduction, thus inhibition of the Shh–Gli1 signaling pathway. Together, our findings show that triptonide effectively inhibits lung cancer cell growth, stemness, and tumorigenicity, and support the notion that triptonide is a new Shh–Gli1 signaling inhibitor and a novel anti-lung cancer drug candidate for further developing effective lung cancer therapeutics.

Increasing evidence shows that numerous cancer-testis antigens (CTAs) are uniquely overexpressed in various types of cancer and most CTAs are oncogenic. Overexpression of oncogenic CTAs promotes carcinogenesis, cancer metastasis, and drug resistance. Oncogenic CTAs are generally associated with poor prognosis in cancer patients and are an important hallmark of cancer, making them a crucial target for cancer immunotherapy. CTAs-targeted antibodies, vaccines, and chimeric antigen receptor-modified T cells (CAR-T) have recently been used in cancer treatment and achieved promising outcomes in the preclinical and early clinical trials. However, the efficacy of current CTA-targeted therapeutics is either moderate or low in cancer therapy. CTA-targeted cancer immunotherapy is facing enormous challenges. Several critical scientific problems need to be resolved: (1) the antigen presentation function of MHC-I protein is usually deficient in cancer patients, so that very low amounts of intracellular CTA epitopes are presented to tumor cell membrane surface, leading to weak immune response and subsequent immunity to CTAs; (2) various immunosuppressive cells are rich in tumor tissues leading to diminished tumor immunity; (3) the tumor tissue microenvironment markedly reduces the efficacy of cancer immunotherapy. In the current review paper, the authors propose new strategies and approaches to overcome the barriers of CTAs-targeted immunotherapy and to develop novel potent immune therapeutics against cancer. Finally, we highlight that the oncogenic CTAs have high tumor specificity and immunogenicity, and are sensible targets for cancer immunotherapy. We predict that CTAs-targeted immunotherapy will bring about breakthroughs in cancer therapy in the near future.

Salvador homolog-1 (SAV1) is a tumor suppressor required for activation of the tumor-suppressive Hippo pathway and inhibition of tumorigenesis. SAV1 is defective in several cancer types. SAV1 deficiency in cells promotes tumorigenesis and cancer metastasis, and is closely associated with poor prognosis for cancer patients. However, investigation of therapeutic strategies to target SAV1 deficiency in cancer is lacking. Here we found that the small molecule lycorine notably increased SAV1 levels in lung cancer cells by inhibiting SAV1 degradation via a ubiquitin–lysosome system, and inducing phosphorylation and activation of the SAV1-interacting protein mammalian Ste20-like 1 (MST1). MST1 activation then caused phosphorylation, ubiquitination, and degradation of the oncogenic Yes-associated protein (YAP), therefore inhibiting YAP-activated transcription of oncogenic genes and tumorigenic AKT and NF-κB signal pathways. Strikingly, treating tumor-bearing xenograft mice with lycorine increased SAV1 levels, and strongly inhibited tumor growth, vasculogenic mimicry, and metastasis. This work indicates that correcting SAV1 deficiency in lung cancer cells is a new strategy for cancer therapy. Our findings provide a new platform for developing novel cancer therapeutics.

Various aggressive cancers, including pancreatic cancer, produce functional blood vessels by neovascularization. Tumor vasculogenic mimicry (VM) promotes cancer progression and is closely associated with the poor prognosis of the cancer patients. Therefore, tumor VM is a sensible target for novel anti-cancer drug discovery. However, there is a lack of effective anti-tumor VM drugs in the clinical setting. In this study, we aim to explore novel agents to effectually inhibit pancreatic cancer cell-mediated tumor VM for anti-cancer therapy. Pancreatic cancer cell lines Patu8988 and Panc1 were utilized as a model. A mouse model was used for in vitro capillary-like structure formation and in vivo Matrigel plug assays to evaluate the anti-tumor VM efficacy of a small molecule triptonide from traditional Chinese herbs. Various methods, including RT-PCR, immunohistochemical staining, and the luciferase gene transcription reporter system, were applied to study the mechanisms of triptonide-exerted anti-tumor VM. Triptonide effectively inhibited pancreatic cancer cell-formed capillary-like structures in vitro and blood vessels in vivo through suppressing pancreatic cancer cell migration, invasion, and VM via inhibiting expression of tumor VM master gene VE-cadherin and pro-migratory gene chemokine C-X-C motif ligand 2 (CXCL2), mainly via reduction of gene promoter activity. Triptonide potently suppresses pancreatic cancer cell-mediated VM by reducing tumor cell migration and invasion and inhibiting expression of VE-cadherin and CXCL2 genes. Our results provide a novel and potent anti-tumor VM drug candidate for further development of effective anti-pancreatic cancer therapy.

Tryptophanyl-tRNA synthetase (TrpRS) in vertebrates contains a N-terminal extension in front of the catalytic core. Proteolytic removal of the N-terminal 93 amino acids gives rise to T2-TrpRS, which has potent anti-angiogenic activity mediated through its extracellular interaction with VE-cadherin. Zinc has been shown to have anti-angiogenic effects and can bind to human TrpRS. However, the connection between zinc and the anti-angiogenic function of TrpRS has not been explored. Here we report that zinc binding can induce structural relaxation in human TrpRS to facilitate the proteolytic generation of a T2-TrpRS-like fragment. The zinc-binding site is likely to be contained within T2-TrpRS, and the zinc-bound conformation of T2-TrpRS is mimicked by mutation H130R. We determined the crystal structure of H130R T2-TrpRS at 2.8 Å resolution, which reveals drastically different conformation from that of wild-type (WT) T2-TrpRS. The conformational change creates larger binding surfaces for VE-cadherin as suggested by molecular dynamic simulations. Surface plasmon resonance analysis indicates more than 50-fold increase in binding affinity of H130R T2-TrpRS for VE-cadherin, compared to WT T2-TrpRS. The enhanced interaction is also confirmed by a cell-based binding analysis. These results suggest that zinc plays an important role in activating TrpRS for angiogenesis regulation.

Lyn is a proto-oncogene overexpressed and constitutively activated in lymphoma, and plays an important role in lymphoma initiation and malignant progression. Hence, the oncogenic Lyn has recently been targeted for novel anti-lymphoma drug discovery; however, the effective Lyn-targeted drug for lymphoma treatment with low toxicity is absent in the clinical setting. The goal of this study is to explore powerful and low toxic Lyn-targeted anti-lymphoma agent. Here we show that triptonide, a small molecule purified from the herb Tripterygium wilfordii Hook F, potently inhibits the proliferation of human B-lymphoma Raji and T-lymphoma Jurkat cells with IC50 of 5.7nM and 4.8nM, respectively. Strikingly, triptonide at a dose of 5mg/kg/day almost completely inhibited the lymphoma growth in human lymphoma cells-xenografted mice without obvious side effects, particularly; the tumors in 6 mice among the 8 xenografted mice were completely eradicated in vivo. Cell biological studies showed that triptonide at the doses of 2.5-10nM notably suppressed B-lymphoma cell colony-forming capability, and that triptonide at the dose of 20nM promoted apoptosis through activation of PARP and caspase 3, but reduction of BCL2 protein levels in the lymphoma cells. Molecular studies revealed that triptonide markedly inhibited oncogenic Lyn transcription through suppressing the promoter activity of the gene, and that it remarkably reduced both total and phosphorylated Lyn proteins, and diminished Lyn downstream ERK and ATK signal pathways. Additionally, triptonide significantly enhanced p38 phosphorylation. Together, triptonide exerts potent anti-lymphoma effect with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn downstream ERK and ATK signal pathways, providing an attractive drug candidate for development of novel anti-lymphoma therapeutics.

Gastric cancer ranks as the third leading cause of cancer-related death worldwide. The uncontrolled tumor growth and robust metastasis are key factors to cause the cancer patient death. Mechanistically, aberrant activation of Notch and NF-κB signaling pathways plays pivotal roles in the initiation and metastasis of gastric cancer. Despite great efforts have been made in recent decades, the effective drug against the advanced and metastatic gastric cancer is still lacking in the clinical setting. In this study, we found that triptonide, a small molecule (MW358) purified from the traditional Chinese medicinal herb Tripterygium wilfordii Hook F, effectively suppressed tumor growth and metastasis in xenograft mice without obvious toxicity at the doses we tested, resulting in potent anti-gastric cancer effect with low toxicity. Triptonide markedly inhibited human metastatic gastric cancer cell migration, invasion, proliferation, and tumorigenicity. Molecular mechanistic studies revealed that triptonide significantly reduced Notch1 protein levels in metastatic gastric cancer cells through degrading the oncogenic protein Notch1 via the ubiquitin-proteasome pathway. Consequently, the levels of Notch1 downstream proteins RBPJ, IKKα, IKKβ were significantly diminished, and nuclear factor-kappa B (NF-κB) phosphorylation was significantly reduced. Together, triptonide effectively suppresses gastric cancer growth and metastasis via inhibition of the oncogenic Notch1 and NF-κB signaling pathways. Our findings provide a new strategy and drug candidate for treatment of the advanced and metastatic gastric cancer.

The mitogen-activated protein kinase (MAPK, 1K) family members ERK, JNK, and p38 play a divergent role in either promoting tumorigenesis or tumor-suppression. Activation of ERK and JNK promotes tumorigenesis; whereas, escalation of p38 inhibits carcinogenesis. As these three MAPK members are controlled by the common up-stream MAPK signaling proteins which consist of MAPK kinases (2K) and MAPK kinase kinases (3K), how to selectively actuate tumor-suppressive p38, not concurrently stimulate tumorigenic ERK and JNK, in cancer cells is a challenge for cancer researchers, and a new opportunity for novel anti-cancer drug discovery. Using human pancreatic cancer cells and xenograft mice as models, we found that a small molecule triptonide first discerningly activated the up-stream MAPK kinase kinase MEKK4, not the other two 3K members ASK1 and GADD45; and then selectively actuated the middle stream MAPK kinase MKK4, not the other two 2K members MKK3 and MKK6; and followed by activation of the MAPK member p38, not the other two members ERK and JNK. These data suggest that triptonide is a selective MEKK4-MKK4-p38 axis agonist. Consequently, selective activation of the MEKK4-MKK4-p38 signaling axis by triptonide activated tumor suppressor p21 and inhibited CDK3 expression, resulting in cancer cell cycle arrest at G2/M phase and marked inhibition of pancreatic cancer cell tumorigenic capability in vitro and tumor growth in xenograft mice. Our findings support the notion that selective activation of tumor-suppressive MEKK4-MKK4-p38-p21signaling pathway by triptonide is a new approach for pancreatic cancer therapy, providing a new drug candidate for development of novel anti-cancer therapeutics.

Metastatic melanoma has a very high mortality rate despite the availability of chemotherapy, radiotherapy, and immunotherapy; therefore, more effective therapeutics are needed. The Hippo pathway plays an inhibitory role in melanoma progression, but the tumor suppressors Salvador homolog-1 (SAV1) and large tumor suppressor 1 (LATS1) in this pathway are down-regulated in melanoma. As a result, the downstream oncogenic Yes-associated protein (YAP) is active, resulting in uncontrolled melanoma growth and metastasis. Therapeutics for remedying SAV1 and LATS1 deficiency in melanoma have not yet been reported in the literature. Here, we show that the small molecule triptonide (MW 358 Da) robustly suppressed melanoma cell tumorigenicity, migration, and invasion. Furthermore, triptonide markedly reduced tumor growth and melanoma lung metastasis in tumor-bearing mice with low toxicity. Molecular mechanistic studies revealed that triptonide promoted SAV1 and LATS1 expression, strongly activated the tumor-suppressive Hippo pathway, degraded oncogenic YAP via the lysosomal pathway, and reduced levels of tumorigenic microphthalmia-associated transcription factor (MITF) in melanoma cells. Triptonide also strongly inhibited activation of AKT, a SAV1-binding signaling protein. Collectively, our results conceptually demonstrate that induction of SAV1 and LATS1 expression and activation of the tumor-suppressive Hippo pathway by triptonide potently inhibits aggressive melanoma cell growth and metastasis. These findings suggest a new strategy for developing therapeutics to treat metastatic melanoma and highlight a novel drug candidate against aggressive melanoma.

F-box proteins, which are subunit recruiting modules of SCF (SKP1-Cullin 1-F-box protein) E3 ligase complexes, play critical roles in the development and progression of human malignancies through governing multiple cellular processes including cell proliferation, apoptosis, invasion and metastasis. Moreover, there are emerging studies that lead to the development of F-box proteins inhibitors with promising therapeutic potential. In this article, we describe how F-box proteins including but not restricted to well-established Fbw7, Skp2 and β-TRCP, are involved in tumorigenesis. However, in-depth investigation is required to further explore the mechanism and the physiological contribution of undetermined F-box proteins in carcinogenesis. Lastly, we suggest that targeting F-box proteins could possibly open new avenues for the treatment and prevention of human cancers.

Gastric cancer is ranked as the third leading cause of cancer-related death in the world. Although extensive efforts have been made in recent decades to treat gastric cancer with various anticancer drugs, effective anti-gastric cancer therapeutics to cure the disease are still lacking in the clinics. Therefore, potent novel anti-gastric cancer drugs are greatly needed. In this study, we explored a novel anti-gastric cancer agent from a medicinal herb named Juncus effusus and found that the active component dehydroeffusol (DHE), a small molecular phenanthrene, effectively inhibited gastric cancer cell proliferation and tumorigenesis by inducing tumor suppressive endoplasmic reticulum (ER) stress and by triggering moderate apoptosis. Mechanistic studies revealed that DHE selectively activated the intracellular tumor suppressive stress response by promoting the overexpression of the key ER stress marker DNA damage-inducible transcript 3 (DDIT3), through upregulation of activating transcription factor 4 (ATF4). Concurrently, DHE suppressed the expression of the cell survival and ER stress marker glucose regulated protein of molecular mass 78 (GRP78) via downregulation of the transcription factor ATF6. In addition, DHE markedly activated the stress response signaling pathway MEKK4-MKK3/6-p38-DDIT3, but significantly inhibited ERK signaling. Our data suggest that DHE inhibits gastric cancer cell growth and tumorigenicity through selectively inducing a robust tumor suppressive ER stress response and a moderate apoptosis response. Therefore, DHE may provide a novel drug candidate for further development of potential anti-gastric cancer therapeutics.

Metastasis is the main cause of death in cancer patients; there is currently no effective treatment for cancer metastasis. This is primarily due to our insufficient understanding of the metastatic mechanisms in cancer. An increasing number of studies have shown that the C-X-C motif chemokine Ligand 12 (CXCL12) is overexpressed in various tissues and organs. It is a key niche factor that nurtures the pre-metastatic niches (tumorigenic soil) and recruits tumor cells (oncogenic “seeds”) to these niches, thereby fostering cancer cell aggression and metastatic capabilities. However, the C-X-C motif chemokine Receptor 4 (CXCR4) is aberrantly overexpressed in various cancer stem/progenitor cells and functions as a CXCL12 receptor. CXCL12 activates CXCR4 as well as multiple downstream multiple tumorigenic signaling pathways, promoting the expression of various oncogenes. Activation of the CXCL12-CXCR4 signaling axis promotes Epithelial-Mesenchymal Transition (EMT) and mobilization of cancer stem/progenitor cells to pre-metastatic niches. It also nurtures cancer cells with high motility, invasion, and dissemination phenotypes, thereby escalating multiple proximal or distal cancer metastasis; this results in poor patient prognosis. Based on this evidence, recent studies have explored either CXCL12- or CXCR4-targeted anti-cancer therapeutics and have achieved promising results in the preclinical trials. Further exploration of this new strategy and its potent therapeutics effect against metastatic cancer through the targeting of the CXCL12- CXCR4 signaling axis may lead to a novel therapy that can clean up the tumor microenvironment (“soil”) and kill the cancer cells, particularly the cancer stem/progenitor cells (“seeds”), in cancer patients. Ultimately, this approach has the potential to effectively treat metastatic cancer.

Metastatic melanoma accounts for 60% of death for skin cancer. Although great efforts have been made to treat the disease, effective drugs against metastatic melanoma still lack at the clinical setting. In the current study, we found that lycorine, a small molecule of isoquinoline alkaloid, significantly suppressed melanoma cell migration and invasion in vitro, and decreased the metastasis of melanoma cells to lung tissues in tumor-bearing mice, resulting in significant prolongation of the survival of the mice without obvious toxicity. Molecular mechanistic studies revealed that lycorine significantly reduced intracellular levels of β-catenin protein through degradation of the protein via the ubiquitin-proteasome pathway, and decreased the expression of β-catenin downstream prometastatic matrix metallopeptidase 9 and Axin2 genes. Collectively, our findings support the notion that targeting the oncogenic β-catenin by lycorine is a new option to inhibit melanoma cell metastasis, providing a good drug candidate potential for development novel therapeutics against metastatic melanoma.

Accumulated data has shown that various vasculogenic tumor cells, including gastric cancer cells, are able to directly form tumor blood vessels via vasculogenic mimicry, supplying oxygen and nutrients to tumors, and facilitating progression and metastasis of malignant tumors. Therefore, tumor vasculogenic mimicry is a rational target for developing novel anticancer therapeutics. However, effective antitumor vasculogenic mimicry-targeting drugs are not clinically available. In this study, we purified 2,7-dihydroxyl-1-methyl-5-vinyl-phenanthrene, termed dehydroeffusol, from the traditional Chinese medicinal herb Juncus effusus L., and found that dehydroeffusol effectively inhibited gastric cancer cell-mediated vasculogenic mimicry in vitro and in vivo with very low toxicity. Dehydroeffusol significantly suppressed gastric cancer cell adhesion, migration, and invasion. Molecular mechanistic studies revealed that dehydroeffusol markedly inhibited the expression of a vasculogenic mimicry master gene VE-cadherin and reduced adherent protein exposure on the cell surface by inhibiting gene promoter activity. In addition, dehydroeffusol significantly decreased the expression of a key vasculogenic gene matrix metalloproteinase 2 (MMP2) in gastric cancer cells, and diminished MMP2 protease activity. Together, our results showed that dehydroeffusol effectively inhibited gastric cancer cell-mediated vasculogenic mimicry with very low toxicity, suggesting that dehydroeffusol is a potential drug candidate for anti-gastric cancer neovascularization and anti-gastric cancer therapy.

A novel impulse laser excitation technique to determine the dynamic response of micro-electro-mechanical systems (MEMS) has been investigated. During the laser excitation experiments, MEMS structures are excited by the wide-band impact force created by the laser–target interaction, and Laser Doppler Vibrometer (LDV) is introduced to measure the vibration velocity of MEMS structures. A distinguishing characteristic of the methodology is that both the excitation and measurement are non-contact, which is especially suitable for the testing of MEMS microstructures that are not easily accessible. This novel excitation method and MEMS modal analysis system are verified by experiments on various cantilever beams. The results show that the laser excitation is capable of exciting the first three modes of cantilevers.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is essential for endothelial cells (ECs) adherens junction and vascular homeostasis; however, the regulatory mechanism of VE-PTP transcription is unknown, and a drug able to promote VE-PTP expression in ECs has not yet been reported in the literature. In this study, we used human ECs as a model to explore small molecule compounds able to promote VE-PTP expression, and found that atorvastatin, a HMG-CoA reductase inhibitor widely used in the clinic to treat hypercholesterolemia-related cardiovascular diseases, strongly promoted VE-PTP transcription in ECs through activating the VE-PTP promoter and upregulating the expression of the transcription factor, specificity protein 1 (SP1). Additionally, atorvastatin markedly reduced VE-cadherin-Y731 phosphorylation induced by cigarette smoke extract and significantly enhanced stability of endothelial adherens junctions. Together, our findings reveal that atorvastatin up-regulates VE-PTP expression, increases VE-cadherin protein levels, and decreases VE-cadherin-Y731 phosphorylation to strengthen EC adherens junctions and maintain vascular cell monolayer integrity, offering a new mechanism of atorvastatin against CSE-induced disruption of vascular integrity and relevant cardio-cerebrovascular disease.

Oncogenic epithelial-mesenchymal transition (oncEMT) plays important roles in the genesis of cancer stem cells (CSCs), malignant tumor initiation and progression, cancer metastasis, and drug resistance. Although the role of oncEMT in tumorigenesis has recently been extensively studied, the initiation of oncEMT is not clearly understood, and its mechanisms of action are still unknown. Emerging evidence suggests that oncEMT is a complex process, which involves multiple endogenous and exogenous factors. Overexpression of several oncogenes and reprogramming factors in precancerous and cancerous cells, including Ras, Myc, Bmi-1, Oct4, Nanog, Slug, Twist, Zeb1, and Zeb2, may initiate oncEMT and tumorigenesis. Defects in key tumor suppressors, such as p53, PTEN, CCN6 protein, and p21 also are associated with oncEMT. MicroRNA (miRNA) may also play a role in the oncEMT. Furthermore, exogenous factors, including chemical carcinogens, viruses, radiation, hypoxia, and acidic microenvironment, can drive oncEMT. Moreover, various growth factors derived from either malignant tumor cells or tumor-associated non-tumor cells in the cancer microenvironment can promote oncEMT. Together, the endogenous and exogenous factors, as well as a hostile cancer microenvironment, initiate the oncEMT program through diverse signaling pathways and networks. However, the dynamic process of initiating oncEMT and the mechanisms are still incompletely understood. Further characterization of the dynamics and mechanisms of the oncEMT will provide new insights into oncogenesis, as well as identify specific oncEMT markers and targets for early diagnosis of cancer and novel anti-cancer drug discovery.

Robust neovascularization and lymphangiogenesis have been found in a variety of aggressive and metastatic tumors. Endothelial sprouting angiogenesis is generally considered to be the major mechanism by which new vasculature forms in tumors. However, increasing evidence shows that tumor vasculature is not solely composed of endothelial cells (ECs). Some tumor cells acquire processes similar to embryonic vasculogenesis and produce new vasculature through vasculogenic mimicry, trans-differentiation of tumor cells into tumor ECs, and tumor cell–EC vascular co-option. In addition, tumor cells secrete various vasculogenic factors that induce sprouting angiogenesis and lymphangiogenesis. Vasculogenic tumor cells actively participate in the formation of vascular cancer stem cell niche and a premetastatic niche. Therefore, tumor cell-mediated neovascularization and lymphangiogenesis are closely associated with tumor progression, cancer metastasis, and poor prognosis. Vasculogenic tumor cells have emerged as key players in tumor neovascularization and lymphangiogenesis and play pivotal roles in tumor progression and cancer metastasis. However, the mechanisms underlying tumor cell-mediated vascularity as they relate to tumor progression and cancer metastasis remain unclear. Increasing data have shown that various intrinsic and extrinsic factors activate oncogenes and vasculogenic genes, enhance vasculogenic signaling pathways, and trigger tumor neovascularization and lymphangiogenesis. Collectively, tumor cells are the instigators of neovascularization. Therefore, targeting vasculogenic tumor cells, genes, and signaling pathways will open new avenues for anti-tumor vasculogenic and metastatic drug discovery. Dual targeting of endothelial sprouting angiogenesis and tumor cell-mediated neovascularization and lymphangiogenesis may overcome current clinical problems with anti-angiogenic therapy, resulting in significantly improved anti-angiogenesis and anti-cancer therapies.

Lycorine is the major active component from the amaryllidaceae family plant Lycoris radiate, a represent traditional Chinese medicinal herb, and is one of the typical alkaloids with pyrrolophenanthridine nucleus core. Lycorine has drawn great interest in medicinal field due to its divergent chemical structures and multiple biological functions, as well as pharmacological effects on various diseases. Accumulated evidence shows that lycorine not only possesses strong pharmacological effects on many diseases, including anti-leukemia, anti-tumor, anti-angiogenesis, anti-virus, anti-bacteria, anti-inflammation, and antimalaria, but also exerts many other biological functions, such as inhibition of acetylcholinesterase and topoisomerase, suppression of ascorbic acid biosynthesis, and control of circadian period length. Notably, lycorine exhibits its numerous pharmacological effects on various diseases with very low toxicity and mild side effects. The divergent chemical structures, multiple biological functions, and very low toxicity of lycorine imply that the agent is a potential drug candidate that warrants for further preclinical and clinic investigation.

Malignant melanoma is the most aggressive form of skin cancer. Although various antimelanoma approaches have been used in the clinics to treat the disease over the last three decades, none of the drugs significantly prolonged the survival of metastatic melanoma patients; hence, effective drugs against metastatic melanoma are highly desired. In this study, we explored an antimetastatic melanoma agent derived from traditional Chinese medicinal herbs and found that jatrorrhizine hydrochloride (JH), an active component of the traditional Chinese medicinal herb Coptis chinensis, inhibited the proliferation and neovascularization of C8161 human metastatic melanoma cells. JH suppressed C8161 cell proliferation in a dose-dependent manner, with a half-maximal inhibitory concentration of 47.4±1.6 μmol/l; however, it did not induce significant cellular apoptosis at doses up to 320 μmol/l. Mechanistic studies showed that JH-induced C8161 cell cycle arrest at the G0/G1 transition, which was accompanied by overexpression of the cell cycle-suppressive genes p21 and p27 at higher doses. Moreover, JH reduced C8161 cell-mediated neovascularization in vitro and in vivo and impeded the expression of the gene for VE-cadherin, a key protein in tumor vasculogenic mimicry and angiogenesis. Taken together, the effective inhibitory effects of JH on metastatic melanoma cell proliferation and neovascularization with low toxicity suggest that JH is a potential new antimelanoma drug candidate.

Cigarette smoking is a major cause of lung cancer. Tumor-associated endothelial cells (TAECs) play important roles in tumor angiogenesis and metastasis. However, whether cigarette smoking can trigger genesis of lung TAECs has not been reported yet. In the current study, we used lung endothelial cell (EC) lines as a model to study the pathological effect of cigarette smoke extracts (CSEs) on human lung ECs, and found that a lower dose of 4% CSEs obviously caused abnormal morphological changes in ECs, increased the permeability of endothelial monolayer, while a higher concentration of 8% CSEs caused EC apoptosis. Strikingly, CSEs induced a 117-fold overexpression of a pro-tumorigenic interleukin-13 receptor α2 gene (IL-13Rα2, also named as CT-19) through activation of the protein kinase A (PKA) and cAMP response element-binding protein (CREB) signaling pathway. A PKA specific inhibitor H89 completely abolished CSEs-induced IL-13Rα2 overexpression. The overexpression of IL-13Rα2 in lung ECs significantly increased the tumorigenic, migratory, and angiogenic capabilities of the cells, suggesting that IL-13Rα2 promotes genesis of lung TAECs. Together, our data show that CSEs activate the PKA, CREB, and IL-13Rα2 axis in lung ECs, and IL-13Rα2 promotes the malignant transformation of lung ECs and genesis of TAECs with robust angiogenic and oncogenic capabilities. Our study provides new insight into the mechanism of CSEs-triggered lung cancer angiogenesis and tumorigenesis, suggesting that the PKA-CREB-IL-13Rα2 axis is a potential target for novel anti-lung tumor angiogenesis and anti-lung cancer drug discovery.

Leukemia cells aberrantly overexpress senescence-suppression telomerase reverse transcriptase (TERT) and down-regulate key senescence-promoting genes to escape complete senescence, resulting in immortalization and malignant progression. Accordingly, induction of complete senescence is a sensible strategy for anti-leukemia therapy. However, effective senescence-based anti-leukemia drugs with low toxicity are currently lacking. In this study, we found that triptonide (chemical name diterpene triepoxide), a small molecule derived from the herb Tripterygium wilfordii Hook, strongly induced complete senescence in cultured acute myeloid leukemia (AML) cell lines, and potently inhibited growth and colony formation of U937 and HL-60 AML cell line with IC50 values of 7.5 and 12nM, respectively. Strikingly, triptonide (4mg/kg) nearly completely suppressed human leukemia cell tumorigenicity (>99%) without obvious toxicity in a mouse xenograft model. Mechanistic studies showed that triptonide induced senescence followed by apoptosis mainly by suppressing transcription of TERT and oncogenic c-Myc, while concomitantly promoting transcription of senescence-promoting genes p16 and p21 and the pro-apoptotic gene encoding DNA damage-inducible transcript 3. These effects of triptonide are mediated by selective mitogen-activated protein kinase kinase-3/p38 signaling pathway activation. Our study provides a conceptual framework for inducing complete senescence as an effective anti-leukemia therapeutic strategy through a "multiple-hits" model and supports further development of triptonide as an anti-cancer agent.

Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery.

We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins (i.e., Tac-β3, Tac-β3Δ741, Tac-β3Δ747, Tac-β3Δ754, Tac-β3Δ759, and Tac-β3ΔNITY) consisting of the extracellular and transmembrane domains of human IL-2 receptor (Tac) and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin αIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3ΔNITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3Δ759, Tac-β3ΔNITY, and Tac-β3Δ754, but not Tac-β3Δ747 or Tac-β3Δ741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3 and Tac-β3ΔNITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3Δ759 and 123/Tac-β3Δ754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3Δ747 and 123/Tac-β3Δ741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.

Melanoma cells actively participate in tumor angiogenesis and vasculogenic mimicry. However, anti-angiogenic therapy in patients with melanoma has not shown a significant survival gain. Thus, new anti-melanoma angiogenic and vasculogenic drugs are highly desired. Using the metastatic melanoma cell line C8161 as a model, we explored melanoma vasculogenic inhibitors and found that lycorine hydrochloride (LH) effectively suppressed C8161 cell-dominant formation of capillary-like tubes in vitro and generation of tumor blood vessels in vivo with low toxicity. Mechanistic studies revealed that LH markedly hindered expression of VE-cadherin in C8161 cells, but did not affect expression of six other important angiogenic and vasculogenic genes. Luciferase assays showed that LH significantly impeded promoter activity of the VE-cadherin gene in a dose-dependent manner. Together, these data suggest that LH inhibits melanoma C8161 cell-dominant vasculogenic mimicry by reducing VE-cadherin gene expression and diminishing cell surface exposure of the protein.

A rapid progress has been made in the field of lymphatic research during the last 15 years. This includes better understanding of the cellular events and molecular players involved in the lymphatic vessel formation and remodeling in development. The key players identified in developmental lymphangiogenesis, including vascular endothelial cell growth factor-C (VEGF-C) / VEGFR-3 and angiopoietins (ANGPTs)/ TIE pathways, are also crucial for pathological lymphatic vessel growth. In solid tumor, tumor cells as well as tumor-associated stromal cells, such as tumor-infiltrating leukocytes, contribute to intra- and peri-tumoral lymphangiogenesis via secreting lymphangiogenic growth factors. Tumor-associated lymphatic endothelial cells also interact actively with tumor cells and leukocytes via secreting various chemokines. It has been well established that tumor lymphangiogenesis promotes tumor cell dissemination to regional lymph nodes. Thus manipulation of lymphangiogenic microenvironment could become another valuable approach in the combat of tumor progression.

Tumor microenvironment (TME) is important in tumor development and may be a target for anti-cancer therapy. The genesis of TME is a dynamic process that is regulated by intrinsic and extrinsic factors and coordinated by multiple genes, cells, and signal pathways. Cancer anaerobic metabolism and various oncogenes may stimulate the genesis of TME. Tumor cells and cancer stem cells actively participate in the genesis of the cancer stem cell niche and tumor neovascularization, important in the initiation of the TME. Various cancer-associated stromal cells, derived niche factors, and tumor-associated macrophages may function as promoters in the genesis of the TME. Dicer1 gene-deleted stromal cells can induce generation of cancer stem cells and initiate tumorigenesis, suggesting that stromal cells also may promote the genesis of the TME. Therefore, the key features of TME include niche-driving oncogenes, cancer anaerobic metabolism, niche-driving cancer stem cells, neovascularization, tumor-associated inflammatory cells, and cancer-associated stromal cells. These features are potential targets for normalization of the malignant TME and effective anti-cancer therapy.

Microbial desulfurization of waste tyre rubber has been investigated with great efforts since 1990s, because waste rubber has created serious ecological and environmental problems. A microbial desulfurization technique for SBR ground rubber has been developed by a novel sulfur-oxidizing bacterium Sphingomonas sp. The adaptability of Sphingomonas sp. with SBR ground rubber was tested with the amounts of SBR ground rubber varying from 0.5 to 4% g/l. The sol fraction of desulfurized SBR ground rubber increased 70%, compared with SBR ground rubber without desulfurization. Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) spectrum and X-ray photoelectron spectroscopy (XPS) analysis of the desulfurized surface of vulcanized SBR flakes revealed that not only the oxidation of crosslinked SS and SC bonds, but also the rupture of CC double bonds had happened to SBR vulcanizates during microbial desulfurization. The cure characteristics, such as scorch time and optimum cure time of natural rubber (NR) vulcanizates filled, were found to decrease with increasing contents of desulfurized SBR ground rubber, due to some reactive groups on its surface. NR vulcanizates filled with desulfurized SBR ground rubber had lower crosslink density and hardness, higher tensile strength and elongation at break, compared with those filled with SBR ground rubber of the same amount. Dynamic mechanical properties indicated that there were better crosslink distribution and stronger interfacial bonding between NR matrix and desulfurized SBR ground rubber. Scanning electron microscope (SEM) photographs showed that the fracture surfaces of NR vulcanizates filled with desulfurized SBR ground rubber had more smooth morphologies. Copyright © 2010 John Wiley & Sons, Ltd.

Epigenetic alterations in stem/progenitor cells play an important role for tumor initiation and development. Although most findings on epigenetic changes in cancers are derived from the established cancers, recent studies have indicated that these changes might be inherited from cancer progenitors. In particular, the epigenetic changes might occur before oncogenetic mutations and foster tumor initiation. Although the aberrant DNA methylation is the main outcome of epigenetic changes in cancer, abnormal RNA interference (RNAi) and chromatin remodeling appear to underlie the changes. A cancer might develop from a tumor-initiating cell (TIC) → precancerous stem cells (pCSCs) → cancer stem cells (CSCs), which might represent various stages of cancer progression. Elucidation of epigenetic changes in these progenitors might be crucial for the discovery of new anti-cancer drugs.

In mammalian cells, specific aminoacyl-transfer RNA (tRNA) synthetases have cytokine functions that require interactions with partners outside of the translation apparatus. Little is known about these interactions and how they facilitate expanded functions that link protein translation to other cellular pathways. For example, an alternative splice fragment of tryptophanyl-tRNA synthetase (TrpRS) and a similar natural proteolytic fragment are potent angiostatic factors that act through the vascular endothelial-cadherin receptor and Akt signaling pathway. Here we demonstrate mobilization of TrpRS for exocytosis from endothelial cells and the potential for plasmin to activate the cytokine function of the extracellular synthetase. Direct physical evidence showed that the annexin II-S100A10 complex, which regulates exocytosis, forms a ternary complex with TrpRS. Functional studies demonstrate that both annexin II and S100A10 regulate trafficking of TrpRS. Thus, complexes of mammalian tRNA synthetases with seemingly disparate proteins may in general be relevant to understanding how their expanded functions are implemented.

MicroRNAs (miRNAs) control the expression of approximately 60% of protein-coding genes and regulate cell metabolism, proliferation, differentiation, and apoptosis. Notably, aberrant expression of miRNAs contributes to several diseases including cancer. Accumulating evidence indicates that miRNAs play important roles in EMT, genesis of cancer stem cells, cancer metabolism and carcinogenesis. Aberrant expression of miRNAs triggers tumor initiation, progression and poor prognosis of cancer patients. Accordingly, oncogenic miRNAs have emerged as diagnostic biomarkers and targets for novel anti-cancer drug discovery. However, the mechanisms of miRNAs contriving tumorigenesis are not completely understood. This review aims to clarify the identification of tumorspecific miRNAs, verification of oncogenic miRNA signatures, and dynamic study of oncogenic miRNAs in cancer initiation and development. Despite sound progress in miRNA-mediated anticancer therapy, several barriers like drug stability, immunogenicity, off-target effects and toxicities still remain. We hope our review could stimulate the further study of miRNAs in cancer research field, which may lead to new insights into the mechanisms of carcinogenesis and create new avenues for targeted cancer therapy.

to explore the relationship between the maxillary sinus volume and the amount of alveolar bone, and the effect of molar loss upon the maxillary sinus was further analyzed,by measuring adult maxillary sinus volume, sinus ridge distance, and calculating the gasification coefficient of maxillary sinus.One hundred and ninety cases (361 maxillary sinus) with CT examinations were collected, they were divided into group A and group B, 121 cases (242 maxillary sinus) of normal subjects served as group A, 42 cases (65 maxillary sinus) with molar part off were B group, in which 31 maxillary sinus with a molar loss were group B1,22 maxillary sinus with two molar loss were B2 group,12 maxillary sinus with three molar loss (one molar remains) were B3 group, 27 cases (54 maxillary sinus) with upper teeth off were C group. Bymeasureing the maxillary sinus volume, sinus ridge distance and the size of the maxillary sinus, calculating the gasification coefficient, we analyzed the relationship between maxillary volume and sinus ridge distance, and comparatively analyzed the differences among the three groups in the size, gasification coefficient, volume of maxillary sinus and sinus ridge distance.In the normal group,the volume of maxillary sinus and sinus ridge distance had a correlation coefficient of -0. 63,(P0.05); Sinus ridge distance in group A was larger than the other two groups (P0.05), and larger in B group than in C group (P0. 05), anteroposterior maxillary sinus diameter and reft-right diameter in C group was greater than in A group and B group(P0.05), group C gasification coeffiecent was less than A group and B group (P0. 05).The volume of maxillary sinus is negatively correlated with the amont of alveolar bone; Upper teeth's shedding promotes maxillary sinus deformation; Maxiuary sinus volume has a tendency to decrease.

Cancer cells have both tumor-adaptive and -suppressive endoplasmic reticulum (ER) stress machineries that determine cell fate. In malignant tumors including lymphoma, constant activation of tumor-adaptive ER stress and concurrent reduction of tumor-suppressive ER stress favors cancer cell proliferation and tumor growth. Current ER stress-based anti-tumor drugs typically activate both tumor-adaptive and -suppressive ER stresses, resulting in low anti-cancer efficacy; hence, selective induction of tumor-suppressive ER stress and inhibition of tumor-adaptive ER stress are new strategies for novel anti-cancer drug discovery. Thus far, specific tumor-suppressive ER stress therapeutics have remained absent in clinical settings. In this study, we explored unique tumor-suppressive ER stress agents from the traditional Chinese medicinal herb Oroxylum indicum, and found that a small molecule oroxin B selectively induced tumor-suppressive ER stress in malignant lymphoma cells, but not in normal cells, effectively inhibited lymphoma growth in vivo, and significantly prolonged overall survival of lymphoma-xenografted mice without obvious toxicity. Mechanistic studies have revealed that the expression of key tumor-adaptive ER-stress gene GRP78 was notably suppressed by oroxin B via down-regulation of up-stream key signaling protein ATF6, while tumor-suppressive ER stress master gene DDIT3 was strikingly activated through activating the MKK3-p38 signaling pathway, correcting the imbalance between tumor-suppressive DDIT3 and tumor-adaptive GRP78 in lymphoma. Together, selective induction of unique tumor-suppressive ER stress and concurrent inhibition of tumor-adaptive ER stress in malignant lymphoma are new and feasible approaches for novel anti-lymphoma drug discovery and anti-lymphoma therapy.

Cachexia or muscle wasting is a common condition that occurs in many chronic diseases. The wasting conditions are characterized by increased levels of TNF-α which was also known as cachectin in the past. But how TNF-α exerts its cachetic effects remains controversial. To clarify this issue, we investigated the impact of TNF-α on C2C12 cell myogenic differentiation. Our results demonstrate that myotube formation was completely inhibited by TNF-α when added to differentiating C2C12 myoblasts. The inhibitory effect of TNF-α on differentiation was accompanied by activation of NF-κB and down regulation of myogenin and Akt. Importantly, TNF-α's effect on differentiation was abolished when IGF-1 was added to the culture. IGF-1 treatment also inhibited NF-κB reporter activity and restored Akt levels. Our data suggest that TNF-α inhibits myogenic differentiation through NF-κB activation and impairment of IGF-1 signaling pathway. The reversal of TNF-α induced inhibition of myogenesis by IGF-1 may have significant therapeutic potential.

Although phospholipid scramblase 1 (PLSCR1) was originally identified based on its capacity to promote transbilayer movement of membrane phospholipids, subsequent studies also provided evidence for its role in cell proliferation, maturation, and apoptosis. In this report, we investigate the potential role of PLSCR1 in leukemic cell differentiation. We show that all-trans retinoic acid (ATRA), an effective differentiation-inducing agent of acute promyelocytic leukemic (APL) cells, can elevate PLSCR1 expression in ATRA-sensitive APL cells NB4 and HL60, but not in maturation-resistant NB4-LR1 cells. ATRA- and phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation is accompanied by increased PLSCR1 expression, whereas only a slight or no elevation of PLSCR1 expression is observed in U937 cells differentiated with dimethyl sulfoxide (DMSO), sodium butyrate, or vitamin D3. Cell differentiation with ATRA and PMA, but not with vitamin D3 or DMSO, results in phosphorylation of protein kinase Cdelta (PKCdelta), and the PKCdelta-specific inhibitor rottlerin nearly eliminates the ATRA- and PMA-induced expression of PLSCR1, while ectopic expression of a constitutively active form of PKCdelta directly increases PLSCR1 expression. Finally, decreasing PLSCR1 expression with small interfering RNA inhibits ATRA/PMA-induced differentiation. Taken together, these results suggest that as a protein induced upon PKCdelta activation, PLSCR1 is required for ATRA- and PMA-triggered leukemic cell differentiation.

Interferons (IFNs) are the principal cytokines responsible for mediating innate immunity against viral infections (7). How IFNs establish an antiviral state in cells has been a subject of investigation since their discovery (21). Nevertheless, mechanisms of IFN action against viral infections remain incompletely understood. IFN antiviral studies have largely focused on several types of IFN-stimulated genes (ISGs), including the double-stranded RNA (dsRNA)-activated protein kinase (PKR), human myxovirus resistance proteins (Mx), 2′,5′-oligoadenylate synthetase (OAS) and its effector protein RNase L, ISG56 (p56), dsRNA-specific adenosine deaminase, and guanylate binding proteins (GBP) (35). Given the critical role of innate immunity in survival from infections, it is not surprising that the antiviral action of IFNs is complex and involves multiple overlapping or related pathways. For instance, mice that are triply deficient for RNase L, PKR, and Mx1 are nevertheless able to mount a substantial, residual IFN antiviral response (48). Therefore, identification of all of the antiviral ISGs is an important step toward a more complete appreciation and understanding of innate immunity. In this regard, within the past several years, global gene expression profiles from IFN-treated cells, obtained by DNA microarrays, have expanded the number of known ISGs from about 33 to >200 (12, 13). Phospholipid scramblase 1 (PLSCR1) is a novel ISG identified as such by way of DNA microarray analysis and confirmed by detailed analysis of the PLSCR1 promoter (12, 49, 50). In fact, PLSCR1 is highly induced by IFN-α, -β, and -γ and also by various growth factors, including epidermal growth factor (EGF), stem cell factor, and granulocyte colony-stimulating factor (30, 51). PLSCR1 is a multiply palmitoylated, lipid-raft-associated endofacial plasma membrane protein, with a proline-rich cytoplasmic domain containing several SH3 and WW domain binding motifs (38). PLSCR1 is proposed to accelerate bidirectional movement of plasma membrane phospholipids during conditions of elevated calcium (50). Transmembrane movement of phospholipids in response to calcium, however, is unaffected by either IFN treatment or PLSCR1 deletion (14, 49, 51). Although the precise biologic function(s) of PLSCR1 and its related isoforms PLSCR2 to 4 remain to be determined (38), recent studies provide intriguing evidence of a role in cell signaling, maturation, and apoptosis. For instance, proliferation and terminal differentiation of certain hematopoietic stem cells (granulocyte precursor) populations is impaired in PLSCR1-null mice (51), and in both monocytic and granulocytic lineages, expression of this protein markedly increases with terminal differentiation into polymorphonuclear leukocytes or macrophages. Conversely, mutations affecting murine PLSCR1 have been associated with a leukemogenic phenotype, which is reversed upon expression of the wild-type (full-length) protein (24, 25). PLSCR1 suppressed ovarian carcinoma in an animal model (37), and elevated expression of PLSCR1 has been shown to be required for normal myeloid differentiation (51). Finally, there is recent evidence that the level of expression of this protein correlates with overall survival in acute myelogenous leukemia (46). PLSCR1 is phosphorylated by select protein kinases, including Abl and Src, tyrosine kinases that participate in multiple growth factor receptor signaling pathways (30, 32, 41). Tyrosine phosphorylation of PLSCR1 by c-Src occurs in response to growth factors such as EGF, resulting in association of phosphorylated PLSCR1 with Shc and the activated EGF receptor complex (30). In the absence of PLSCR1, the activation of c-Src kinase through EGF receptor (and related receptors) is markedly attenuated, suggesting that PLSCR1 plays a role in growth factor-dependent recruitment or activation of c-Src kinase, potentially through its interaction in membrane lipid rafts (30, 40). Palmitoylation of PLSCR1 is required for insertion into the plasma membrane (44). However, when palmitoylation does not occur, the importin α/β nucleopore transport system has recently been shown to import PLSCR1 into the nucleus where it binds DNA (6, 44). Accordingly, newly synthesized PLSCR1 appeared in nuclei after IFN induction of PLSCR1 in the human ovarian carcinoma cell line, Hey1B (44). PLSCR1 is the only member of the PLSCR family thus far shown to be inducible by IFNs. These findings raise the possibility that PLSCR1 may contribute to the antiviral effects of IFNs by affecting viral penetration, IFN-stimulated cell signaling pathways at the plasma membrane, the transcription of antiviral genes in the nucleus, and/or by directly blocking specific stages in the viral replication cycle. To determine the involvement of PLSCR1 in the IFN-induced antiviral state, we have compared viral replication in wild-type and PLSCR1−/− mouse cells as well as in human cells in which PLSCR1 levels were decreased with short interfering RNA (siRNA). Our results demonstrate a marked suppression of viral replication by PLSCR1 which is accompanied by the enhanced expression of a specific subset of antiviral ISGs.

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1-/- embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.

Malignant tumors aberrantly overexpress various embryonic genes and proto-oncogenes, including a variety of cancer-testis antigens (CTAs). CTAs belong to a class of testis-derived proteins which are only expressed in germ cells in the male testis, and the expression of CTA genes is entirely silenced in the adult somatic tissues. They are, however, aberrantly overexpressed in a variety of malignant tumor tissues. Emerging evidence shows that a number of CTAs promote epithelialmesenchymal transition (EMT) and genesis of cancer stem like cells, escalating tumorigenesis, invasion, and metastasis. The can cer-testis antigens, such as SSX, MAGE-D4B, CAGE, piwil2, and CT45A1, upregulate EMT and metastatic genes, promoting EMT and tumor dissemination. In addition, certain members of CTAs, including Piwil2, DNAJB8, CT45A1, MAGE-A, GAGE, and SPANX, are implicated in the initiation or maintenance, of cancer stem-like cells, promoting tumorigenesis and malignant progression. Clinically CTAs are closely associated with poor prognosis in cancer patients. Intriguely, CTAs are strongly immunogenic and normally restricted to the male testis after birth, however, these proteins are aberrantly overexpressed in cancer stem-like cells and in a variety of cancers, suggesting their target potential for cancer immunotherapy, as diagnostic biomarkers, and as targets for novel anticancer drug discovery. Thus, the targeting of tumorigenic CTAs is a promising strategy to eradicate cancer stem-like cells and inhibit tumorigenesis for effective cancer treatment.