mRNA-Engineered CD5-CAR-γδTCD5- Cells for the Immunotherapy of T-Cell Acute Lymphoblastic Leukemia.

Clinical trials of Chimeric Antigen Receptor T-cell (CAR-T) therapy have demonstrated remarkable success in treating both solid tumors and hematological malignancies. Nanobodies (Nbs) have emerged as promising antigen-targeting domains for CARs, owing to their high specificity, robust stability, and strong affinity, leading to significant advancements in the field of Nb-CAR-T. In the realm of T-cell acute lymphoblastic leukemia (T-ALL) targets, CD5 stands out as a potentially excellent candidate for T-cell-based CAR therapy, due to its distinct expression on the surface of malignant T-ALL cells. To mitigate graft-versus-host disease associated with allogeneic CAR-T, γδT cells are selected and stimulated from peripheral blood mononuclear cells, and γδT cells are engineered via CRISPR/Cas9 to eliminate fratricide, enabling the creation of fratricide-resistant CAR-γδTCD5- cells. In vitro transcribed (IVT) mRNA is used to construct CAR-T, presenting a safer, faster, and cost-effective method compared to traditional viral vector approaches. In this study, a CD5-VHH library is constructed, and specific CD5-nanobodies are screened for subsequent use in CD5-CAR-γδTCD5- therapy. IVT-mRNA-CD5-CAR-γδTCD5- cells exhibited favorable functional characteristics and demonstrated antitumor efficacy against malignant T cell lines, underlining the potential for advancing mRNA-CD5-CAR-γδTCD5- therapy.

WP1066, a small molecule inhibitor of STAT3, chemosensitizes paclitaxel-resistant ovarian cancer cells to paclitaxel by simultaneously inhibiting the activity of STAT3 and the interaction of STAT3 with Stathmin.

Paclitaxel is widely used to treat cancer, however, drug resistance limits its clinical utility. STAT3 is constitutively activated in some cancers, and contributes to chemotherapy resistance. Currently, several STAT3 inhibitors including WP1066 are used in cancer clinical trials. However, whether WP1066 reverses paclitaxel resistance and the mechanismremains unknown. Here, we report that in contrast to paclitaxel-sensitive parental cells, the expressions of several pro-survival BCL2 family members such as BCL-2, BCL-XL and MCL-1 are higher in paclitaxel-resistant ovarian cancer cells. Meanwhile, STAT3 is constitutively activated while stathmin loses its activity in paclitaxel-resistant cells. Importantly, WP1066 amplifies the inhibition of cell proliferation, colony-forming ability and apoptosis of ovarian cancer cells induced by paclitaxel. Mechanistically, WP1066, on the one hand, interferes the STAT3/Stathmin interaction, causing unleash of STAT3/Stathmin from microtubule, thus destroying microtubule stability. This process results in reduction of Ac-α-tubulin, further causing MCL-1 reduction. On the other hand, WP1066 inhibits phosphorylation of STAT3 by JAK2, and blocks its nuclear translocation, therefore repressing the transcription of pro-survival targets such as BCL-2, BCL-XL and MCL-1. Finally, the two pathways jointly promote cell death. Our findings reveal a new mechanism wherein WP1066 reverses paclitaxel-resistance of ovarian cancer cells by dually inhibiting STAT3 activity and STAT3/Stathmin interaction, which may layfoundation for WP1066 combined with paclitaxel in treating paclitaxel-resistant ovarian cancer.

Abrogation of ATR function preferentially augments cisplatin-induced cytotoxicity in PTEN-deficient breast cancer cells.

Targeting replication stress response is currently emerging as new therapeutic strategy for cancer treatment, based on monotherapy and combination approaches. As a key sensor in response to DNA damage, ataxia telangiectasia and rad3-related (ATR) kinase has become a potential therapeutic target as tumor cells are to rely heavily on ATR for survival. The tumor suppressor phosphatase and tensin homolog (PTEN) plays a crucial role in maintaining chromosome integrity. Although ATR inhibition was recently confirmed to show a synergistic inhibitory effect in PTEN-deficient triple-negative breast cancer cells, the molecular mechanism needs to be further elucidated. Additionally, whether the PTEN-deficient breast cancer cells are more preferentially sensitized than PTEN-wild type breast cancer cells to cisplatin plus ATR inhibitor remains unanswered. We demonstrate PTEN dysfunction promotes the killing effect of ATR blockade through the use of RNA interference for PTEN and a highly selective ATR inhibitor VE-821, and certify that VE-821 (1.0 µmol/L) aggravates cytotoxicity of cisplatin on breast cancer cells, especially PTEN-null MDA-MB-468 cells which show more chemoresistance than PTEN-expressing MDA-MB-231 cells. The co-treatment with VE-821 and cisplatin significantly reduced cell viability and proliferative capacity compared with cisplatin mono-treatment (P < 0.05). The increased cytotoxic activity is tied to the enhanced poly (ADP-ribose) polymerase (PARP) cleavage and consequently cell death due to the decrease in phosphorylation levels of checkpoint kinases 1 and 2 (CHK1/2), the reduction of radiation sensitive 51 (RAD51) foci and the increase in phosphorylation of the histone variant H2AX (γ-H2AX) foci (P < 0.05) as well. Together, these findings suggest combination therapy of ATR inhibitor and cisplatin may offer a potential therapeutic strategy for breast tumors.

Evaluation of B7-H3 Targeted Immunotherapy in a 3D Organoid Model of Craniopharyngioma.

A craniopharyngioma (CP) is a rare epithelial tumor of the sellar and parasellar region. CPs are difficult to treat due to their anatomical proximity to critical nervous structures, which limits the ability of the surgeon to completely resect the lesion, exposing patients to a high risk of recurrence. The treatment of craniopharyngiomas is primarily surgery and radiotherapy. So far, neither a cell line nor an animal model has been established, and thus data on other treatment options, such as chemotherapy and immunotherapy, are limited. Here, the expression profile of the pan-cancer antigen B7-H3 in various cancer types including CP was examined by immunohistochemistry. An in vitro organoid model was established by using fresh tissue biospecimens of CP. Based on the organoid model, we evaluated the antitumor efficacy of B7-H3-targeted immunotherapy on CP. As a result, the highest expression of B7-H3 was observed in CP tissues across various cancer types. Although B7-H3-targeted chimeric antigen-receptor T cells show obvious tumor-killing effects in the traditional 2D cell culture model, limited antitumor effects were observed in the 3D organoid model. The B7-H3-targeted antibody-DM1 conjugate exhibited a potent tumor suppression function both in 2D and 3D models. In conclusion, for the first time, we established an organoid model for CP and our results support that B7-H3 might serve as a promising target for antibody-drug conjugate therapy against craniopharyngioma.

PUMA overexpression dissociates thioredoxin from ASK1 to activate the JNK/BCL-2/BCL-XL pathway augmenting apoptosis in ovarian cancer.

ASK1-JNK signaling promotes mitochondrial dysfunction-mediated apoptosis, but the bridge between JNK and apoptosis is not fully understood. PUMA induces apoptosis through BAX/BAK. Our previous study suggests a therapeutic potential of PUMA for ovarian cancer. However, whether and how PUMA activates ASK1 remains unclear. Here, we found for the first time that PUMA activated ASK1 by dissociating thioredoxin (TRX) from ASK1, however, it neither interacted with ASK1 nor TRX. Furthermore, PUMA overexpression caused ROS release from mitochondrial. H2O2 significantly impaired the interaction of ASK1 with TRX, whereas ROS scavenger NAC effectively abrogated the H2O2 effect, partly rescued PUMA-interfered interaction of ASK1 with TRX, and also abolished ASK1 phosphorylation. Interestingly, PUMA could not impair the association of ASK1 with TRX-C32S or TRX-C35S, two TRX mutants which are no longer oxidized in response to ROS. We further showed that PUMA activated ASK1-JNK axis to phosphorylate BCL-2 and BCL-XL, further augmenting apoptosis of ovarian cancer cells. In vivo, PUMA adenovirus combined with paclitaxel significantly inhibited intrinsically cisplatin-resistant ovarian cancer growth, and caused phosphorylation of BCL-2 and BCL-XL. Our results from human ovarian cancer TMA chips also revealed a positive correlation between PUMA expression and the phosphorylation of BCL-2 and BCL-XL. More importantly, all patients had no distal metastasis, implying a possibly clinical significance. Collectively, our results reveal a new pro-apoptotic signal amplification mechanism for PUMA by which PUMA overexpression first induces ROS-mediated dissociation of TRX from ASK1, and then causes JNK activation-triggering BCL-2/BCL-XL phosphorylation, ultimately augmenting apoptosis in ovarian cancer.

Genome-scale CRISPR-Cas9 screen reveals novel regulators of B7-H3 in tumor cells.

BACKGROUND: Despite advances in B7 homolog 3 protein (B7-H3) based immunotherapy, the development of drug resistance remains a major clinical concern. The heterogeneity and emerging loss of B7-H3 expression are the main causes of drug resistance and treatment failure in targeted therapies, which reveals an urgent need to elucidate the mechanism underlying the regulation of B7-H3 expression. In this study, we identified and explored the crucial role of the transcription factor SPT20 homolog (SP20H) in B7-H3 expression and tumor progression. METHODS: Here, we performed CRISPR/Cas9-based genome scale loss-of-function screening to identify regulators of B7-H3 in human ovarian cancer cells. Signaling pathways altered by SP20H knockout were revealed by RNA sequencing. The regulatory role and mechanism of SP20H in B7-H3 expression were validated using loss-of-function and gain-of-function assays in vitro. The effects of inhibiting SP20H on tumor growth and efficacy of anti-B7-H3 treatment were evaluated in tumor-bearing mice. RESULTS: We identified SUPT20H (SP20H) as negative and eIF4E as positive regulators of B7-H3 expression in various cancer cells. Furthermore, we provided evidence that either SP20H loss or TNF-α stimulation in tumor cells constitutively activates p38 MAPK-eIF4E signaling, thereby upregulating B7-H3 expression. Loss of SP20H upregulated B7-H3 expression both in vitro and in vivo. Additionally, deletion of SP20H significantly suppressed tumor growth and increased immune cells infiltration in tumor microenvironment. More importantly, antibody-drug conjugates targeting B7-H3 exhibited superior antitumor performance against SP20H-deficient tumors relative to control groups. CONCLUSIONS: Activation of p38 MAPK-eIF4E signaling serves as a key event in the transcription initiation and B7-H3 protein expression in tumor cells. Genetically targeting SP20H upregulates target antigen expression and sensitizes tumors to anti-B7-H3 treatment. Collectively, our findings provide new insight into the mechanisms underlying B7-H3 expression and introduce a potential synergistic target for existing antibody-based targeted therapy against B7-H3.

JNK initiates Beclin-1 dependent autophagic cell death against Akt activation.

ABT-199, a specific inhibitor of the Bcl-2 protein, is widely used in clinical trials for hematological tumors and rarely applied to the research of solid tumors. In this study, we used Bax/Bak double knockout (KO) and knockdown (KD) cells as the model and found that ABT-199 initiated autophagic cell death independent of Bax and Bak. ABT-199 initiated Beclin-1-dependent autophagy, which led to cell death. Furthermore, inactivated Akt released Beclin-1 from the 14-3-3 protein through a change in the phosphorylation state of Beclin-1 in ABT-199-treated cells. Moreover, JNK antagonized the function of Akt in Beclin-1-mediated autophagy by phosphorylating the 14-3-3 protein. Phosphorylated 14-3-3 exhibited a decreased interaction with Beclin-1. Therefore, ABT-199 activated the JNK-Akt-14-3-3 signaling pathway to mediate the Beclin-1-dependent autophagic death of Bax/Bak KO and KD cells. These findings may extend the therapeutic application of ABT-199 to colon cancer, particularly apoptosis-deficient tumors.

Intercellular Adhesion Molecule-1 as Target for CAR-T-Cell Therapy of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) comprises lethal malignancies with limited treatment options. Chimeric antigen receptor T (CAR-T) cell therapy is an effective immunotherapeutic strategy that has demonstrated unprecedented efficacy in the treatment of hematological malignancies but has shown limited success in the management of some solid tumors. Many malignant tumors are related to increased expression of intercellular adhesion molecule-1 (ICAM1), providing a rationale for ICAM1-specific immunotherapies for the treatment of cancer. Here, we validated the expression of ICAM1 in TNBC tissues. Subsequently, we generated a phage-displayed single-chain variable fragment (scFv) library using splenocytes from ICAM1-immunized mice and selected a novel ICAM1-specific scFv, mG2-scFv. Using mG2-scFv as the extracellular antigen binding domain, we constructed ICAM1-specific CAR-T cells and demonstrated the robust and specific killing of TNBC cell lines in vitro. Most importantly, in the TNBC mouse model, ICAM1-specific CAR-T cells significantly reduced the growth of the TNBC tumor, resulting in long-term remission and improved survival. Together, these results indicated that ICAM1-specific CAR-T cells have high therapeutic potential against ICAM1-positive TNBC tumors.

Diosgenin exhibits tumor suppressive function via down-regulation of EZH2 in pancreatic cancer cells.

Pancreatic cancer (PC) is one of the most aggressive and lethal malignancies worldwide. Although significant progress has been made in oncology treatment, this refractory disease is still become intractable. Natural herb product diosgenin is described to exhibit vast range of pharmacological activities in preclinical studies, including anti-cancer activities. Accumulating data demonstrated that Enhancer of zeste homolog 2 (EZH2) as an oncogenic protein is over-expressed in various human cancers, including PC. However, the underlying mechanism has not been fully understood. In this study, we aim to investigate the anti-cancer properties and molecular basis of diosgenin in PC cells. Significant inhibition of cell proliferation was observed in diosgenin treated Patu8988 and Panc-1 cells in a dose- and time-dependent manner. Apoptotic cell death and G2/M phase arrest were also induced by diosgenin treatment in PC cells. Moreover, obvious inhibition of cell migration and invasive capacities was detected in diosgenin treated PC cells. Mechanistically, the expression levels of EZH2 and its target Vimentin were reduced, and PTEN was promoted after diosgenin exposure. Our results further supported that EZH2 signaling was closely associated with the anti-tumor characteristics of diosgenin in PC cells. Therefore, inhibition of EZH2 by diosgenin could be a promising therapeutic method for PC treatment.

Frequent B7-H3 overexpression in craniopharyngioma.

Craniopharyngiomas (CPs) are uncommon intracranial benign neoplasms that located in sellar/parasellar region with clinically challenging. B7-H3 is an immune checkpoint molecule highly expressed in many malignant tumors. In this study, we analyzed whether B7-H3 is expressed in 44 CPs samples (adamantinomatous CPs: n = 30 and papillary CPs: n = 14), and whether it could serve as an immunotherapy target in CPs. Immunohistochemical analysis showed that B7-H3 was highly expressed in adamantinomatous CPs (184.3 ±â€¯13.58) and papillary CPs (223.2 ±â€¯11.89), while almost undetectable in normal brain tissue (24 ±â€¯4.9). Besides, B7-H3 expression level was correlated with poor prognosis of patients with CPs. Immunofluorescence and Western blot analysis further suggested that ß-catenin co-localized with B7-H3 and could promote its expression in adaCPs. B7-H3 expression level was positively correlated with staining intensity of IBA1+ cells, but negatively with T cell infiltration in CPs, suggesting that B7-H3 might play a role in the regulation of tumor microenvironment in CPs. Moreover, B7-H3/CD3 bi-specific T cell engager (BiTE) efficiently inhibited the growth of human primary craniopharyngioma cells in a time- and dose-dependent manner. Our results revealed B7-H3 was highly expressed in CPs and targeting B7-H3 might therefore be an effective therapeutic strategy against craniopharyngioma.

Correction: Guanylate-binding protein 2 regulates Drp1-mediated mitochondrial fission to suppress breast cancer cell invasion.

The work was supported by the Natural Science Foundation of China (NSFC)-81773188, 81760557, 81703081, and Program for New Century Excellent Talents in University (NCET)-12-0381. And we would like to appreciate the help given to the work by the lab of Prof. Fuyu Yang's from Institute of Biophysics and Prof. Yaping Tu from Creighton University in USA.

Structural and functional insights into the regulation of the lysis-lysogeny decision in viral communities.

Communication is vital for all organisms including microorganisms, which is clearly demonstrated by the bacterial quorum-sensing system. However, the molecular mechanisms underlying communication among viruses (phages) via the quorum-sensing-like 'arbitrium' system remain unclear. Viral or host densities are known to be related to an increased prevalence of lysogeny; however, how the switch from the lytic to the lysogenic pathway occurs is unknown. Thus, we sought to reveal mechanisms of communication among viruses and determine the lysogenic dynamics involved. Structural and functional analyses of the phage-derived SAIRGA and GMPRGA peptides and their corresponding receptors, phAimR and spAimR, indicated that SAIRGA directs the lysis-lysogeny decision of phi3T by modulating conformational changes in phAimR, whereas GMPRGA regulates the lysis-lysogeny pathway by stabilizing spAimR in the dimeric state. Although temperate viruses are thought to share a similar lytic-lysogenic cycle switch model, our study suggests the existence of alternative strain-specific mechanisms that regulate the lysis-lysogeny decision. Collectively, these findings provide insights into the molecular mechanisms underlying communication among viruses, offering theoretical applications for the treatment of infectious viral diseases.

ARD1 contributes to IKKß-mediated breast cancer tumorigenesis.

The expression of IκB kinase ß (IKKß) promotes the growth of breast cancer cells. Meanwhile, IKKß mediates the phosphorylation and subsequent degradation of arrest-defective protein 1 (ARD1). However, the relationship between IKKß and ARD1 in the occurrence of breast cancer has not been reported. In this study, we found that IKKß not only acts directly on mammalian target of rapamycin (mTOR) activity but also indirectly acts on mTOR activity through posttranscriptional modification of ARD1, thereby effectively promoting the growth of breast cancer cells. ARD1 prevents mTOR activity and breast cancer cell growth by stabilizing tuberous sclerosis complex 2 (TSC2) to induce autophagy. Moreover, acetylation of heat shock protein 70 (Hsp70) also contributes to ARD1-mediated autophagy. Therefore, upstream IKKß can further promote the occurrence of breast cancer by mediating the function of ARD1.

Combination of DESI2 and endostatin gene therapy significantly improves antitumor efficacy by accumulating DNA lesions, inducing apoptosis and inhibiting angiogenesis.

DESI2 is a novel pro-apoptotic gene. We previously reported that DESI2 overexpression induces S phase arrest and apoptosis by activating checkpoint kinases. This work was to test whether the combination of endostatin, an endogenous antiangiogenic inhibitor, with DESI2 could improve the therapy efficacy in vitro and in vivo. The recombinant plasmid co-expressing DESI2 and endostatin was encapsulated with DOTAP/Cholesterol cationic liposome. Mice bearing CT26 colon carcinoma and LL2 lung cancer were treated with the DNA-liposome complex. We found that, in vitro, the combination of DESI2 and endostatin more efficiently inhibited proliferation of CT26, LL2, HCT116 and A549 cancer cells via apoptosis, as assessed by MTT assay, colony-formation assays, flow cytometric analysis, hoechst staining and activation of caspase-3, respectively. In addition, DESI2 overexpression caused up-regulation of RPS7, a substrate of DESI2 deubiquitination. Furthermore, siRNA targeting RPS7 partially abrogated, whereas RPS7 overexpression enhanced DESI2-induced inhibition of cell proliferation. Importantly, the combination also caused DNA lesions accumulation, which further promotes apoptosis. Mechanistic rationale suggested that endostatin first inhibits DNA-PKcs kinase, and partly abrogated DESI2-induced phosphorylation of DNA-PKcs, leading to increase of DNA damage, then contributes to DESI2-induced apoptosis. In vivo, the combined gene therapy more significantly inhibited tumor growth and efficiently prolonged the survival of tumor bearing mice than mono therapy. The improved antitumor effect was associated with inhibition of cell proliferation via apoptosis, as analyzed by TUNEL assay and PCNA immunostaining. The combination also inhibited angiogenesis, as assessed by alginate-encapsulated tumor cell assay and CD31 staining. Our data suggest that the combined gene therapy of DESI2 and endostatin can significantly enhance the antitumor activity as a DNA lesions accumulator, apoptosis inducer and angiogenesis inhibitor. The present study may provide a novel method for the treatment of cancer.

SKLB-23bb, A HDAC6-Selective Inhibitor, Exhibits Superior and Broad-Spectrum Antitumor Activity via Additionally Targeting Microtubules.

Our previous study reported that SKLB-23bb, an orally bioavailable HDAC6-selective inhibitor, exhibited superior antitumor efficiency both in vitro and in vivo in comparison with ACY1215, a HDAC6-selective inhibitor recently in phase II clinical trial. This study focused on the mechanism related to the activity of SKLB-23bb. We discovered that despite having HDAC6-selective inhibition equal to ACY1215, SKLB-23bb showed cytotoxic effects against a panel of solid and hematologic tumor cell lines at the low submicromolar level. Interestingly, in contrast to the reported HDAC6-selective inhibitors, SKLB-23bb was more efficient against solid tumor cells. Utilizing HDAC6 stably knockout cell lines constructed by CRISPR-Cas9 gene editing, we illustrated that SKLB-23bb could remain cytotoxic independent of HDAC6 status. Investigation of the mechanism confirmed that SKLB-23bb exerted its cytotoxic activity by additionally targeting microtubules. SKLB-23bb could bind to the colchicine site in ß-tubulin and act as a microtubule polymerization inhibitor. Consistent with its microtubule-disrupting ability, SKLB-23bb also blocked tumor cell cycle at G2-M phase and triggered cellular apoptosis. In solid tumor xenografts, oral administration of SKLB-23bb efficiently inhibited tumor growth. These results suggested that SKLB-23bb was an orally bioavailable HDAC6 and microtubule dual targeting agent. The microtubule targeting profile enhanced the antitumor activity and expanded the antitumor spectrum of SKLB-23bb, thus breaking through the limitation of HDAC6 inhibitors. Mol Cancer Ther; 17(4); 763-75. ©2018 AACR.

Long non-coding RNA SPRY4-IT1 promotes cell proliferation and invasion by regulation of Cdc20 in pancreatic cancer cells.

Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) play a critical role in the development of human cancers including pancreatic cancer. Long non-coding RNA SPRY4-IT1 (sprouty4-intron transcript 1) has been reported to play an oncogenic role in various types of human carcinomas. However, the role of SPRY4-IT1 in pancreatic cancer is unclear. The objective of this study was to determine the function of SPRY4-IT1 on proliferation and invasion in pancreatic cancer. In the current study, we dissected the function and mechanism of SPRY4-IT1 by multiple approaches including Real-time RT-PCR, Western blotting analysis, MTT assay, Wound healing assay, Transwell assay, and transfection. We found that down-regulation of SPRY4-IT1 inhibited cell growth and induced cell apoptosis in pancreatic cancer cells. Moreover, SPRY4-IT1 knockdown induced cell cycle arrest at G0/G1 phase. Furthermore, inhibition of SPRY4-IT1 retarded cell migration and invasion in pancreatic cancer cells. Overexpression of SPRY4-IT1 enhanced cell growth and invasion, and inhibited cell apoptosis in pancreatic cancer cells. Mechanistically, suppression of SPRY4-IT1 inhibited the expression of Cdc20 in pancreatic cancer cells. Our findings demonstrated that inhibition of SPRY4-IT1 could be a potential therapeutic approach for the treatment of pancreatic cancer.

PP2A mediates apoptosis or autophagic cell death in multiple myeloma cell lines.

The crosstalk between apoptosis and autophagy contributes to tumorigenesis and cancer therapy. The process by which BetA (betulinic acid), a naturally occurring triterpenoid, regulates apoptosis and autophagy as a cancer therapy is unclear. In this study, we show for the first time that protein phosphatase 2A (PP2A) acts as a switch to regulate apoptosis and autophagic cell death mediated by BetA. Under normal conditions, caspase-3 is activated by the mitochondrial pathway upon BetA treatment. Activated caspase-3 cleaves the A subunit of PP2A (PP2A/A), resulting in the association of PP2A and Akt. This association inactivates Akt to initiate apoptosis. Overexpression of Bcl-2 attenuates the mitochondrial apoptosis pathway, resulting in caspase-3 inactivation and the dissociation of PP2A and Akt. PP2A isolated from Akt binds with DAPK to induce autophagic cell death. Meanwhile, in vivo tumor experiments have demonstrated that BetA initiates different types of cell death in a myeloma xenograft model. Thus, PP2A can shift myeloma cells from apoptosis to autophagic cell death. These findings have important implications for the therapeutic application of BetA, particularly against apoptosis-resistant cancers.

Guanylate-binding protein 2 regulates Drp1-mediated mitochondrial fission to suppress breast cancer cell invasion.

Guanylate-binding protein 2 (GBP2) is a member of the large GTPase superfamily that is strongly induced by interferon-γ (IFN-γ). Although the biochemical characteristics of GBP2 have been reported in detail, its biological function has not been thoroughly elucidated to date. To the best of our knowledge, this study presents the first demonstration that GBP2 inhibits mitochondrial fission and cell metastasis in breast cancer cells both in vitro and in vivo. Our previous work demonstrated that dynamin-related protein 1 (Drp1)-dependent mitochondrial fission has a key role in breast cancer cell invasion. In this study, we demonstrate that GBP2 binds directly to Drp1. Elimination of Drp1 by shRNA or Mdivi-1 (a Drp1-specific inhibitor) suppressed GBP2's regulatory function. Furthermore, GBP2 blocks Drp1 translocation from the cytosol to mitochondria, thereby attenuating Drp1-dependent mitochondrial fission and breast cancer cell invasion. In summary, our data provide new insights into the function and molecular mechanisms underlying GBP2's regulation of breast cancer cell invasion.

Combination of DESI2 and IP10 gene therapy significantly improves therapeutic efficacy against murine carcinoma.

DESI2 (also known as PNAS-4) is a novel pro-apoptotic gene activated during the early response to DNA damage. We previously reported that overexpression of DESI2 induces S phase arrest and apoptosis by activating checkpoint kinases. The present study was designed to test whether combination of DESI2 and IP10 could improve the therapy efficacy in vitro and in vivo. The recombinant plasmid co-expressing DESI2 and IP10 was encapsulated with DOTAP/Cholesterol nanoparticle. Immunocompetent mice bearing CT26 colon carcinoma and LL2 lung cancer were treated with the complex. We found that, in vitro, the combination of DESI2 and IP10 more efficiently inhibited proliferation of CT26, LL2, SKOV3 and A549 cancer cells via apoptosis. In vivo, the combined gene therapy more significantly inhibited tumor growth and efficiently prolonged the survival of tumor bearing mice. Mechanistically, the augmented antitumor activity in vivo was associated with induction of apoptosis and inhibition of angiogenesis. The anti-angiogenesis was further mimicked by inhibiting proliferation of immortalized HUVEC cells in vitro. Meanwhile, the infiltration of lymphocytes also contributed to the enhanced antitumor effects. Depletion of CD8+ T lymphocytes significantly abrogated the antitumor activity, whereas depletion of CD4+ T cells or NK cells showed partial abrogation. Our data suggest that the combined gene therapy of DESI2 and IP10 can significantly enhance the antitumor activity as apoptosis inducer, angiogenesis inhibitor and immune response initiator. The present study may provide a novel and effective method for treating cancer.

The role of ROS and subsequent DNA-damage response in PUMA-induced apoptosis of ovarian cancer cells.

PUMA is a member of the "BH3-only" branch of the BCL-2 family. Our previous study suggests a therapeutic potential of PUMA in treating ovarian cancer, however, the action mechanism of PUMA remains elusive. In this work, we found that in PUMA adenovirus-infected A2780s ovarian cancer cells, exogenous PUMA was partially accumulated in the cytosol and mainly located to the mitochondria. We further showed that PUMA induces mitochondrial dysfunction-mediated apoptosis and ROS generation through functional BAX in a ROS generating enzyme- and caspase-independent manner irrespective of their p53 status, and results in activation of Nrf2/HO-1 pathway. Furthermore, PUMA induces DNA breaks in γ-H2AX staining, and causes activation of DNA damage-related kinases including ATM, ATR, DNA-PKcs, Chk1 and Chk2, which are correlated with the apoptosis. PUMA also results in ROS-triggered JNK activation. Intriguingly, JNK plays a dual role in both DNA damage response and apoptosis, and has an additional contribution to apoptosis. Taken together, we have provided new insight into the action mechanism by which elevated PUMA first induces ROS generation then results in DNA damage response and JNK activation, ultimately contributing to apoptosis in ovarian cancer cells.