Kmt2a cooperates with menin to suppress tumorigenesis in mouse pancreatic islets

The reported incidence of pancreatic neuroendocrine tumors (PanNETs) has increased, due in large part to improvements in detection and awareness. However, therapeutic options are limited and a critical need exists for understanding a more thorough characterization of the molecular pathology underlying this disease. The Men1 knockout mouse model recapitulates the early stage of human PanNET development and can serve as a foundation for the development of advanced mouse models that are necessary for preclinical testing. Menin, the product of the MEN1 gene, has been shown to physically interact with the KMT2A and KMT2B histone methyltransferases. Both the KMT2A and MEN1 genes are located on chromosome 11q, which frequently undergoes loss of heterozygosity (LOH) in PanNETs. We report herein that inactivation of Kmt2a in Men1-deficient mice accelerated pancreatic islet tumorigenesis and shortened the average life span. Increases in cell proliferation were observed in mouse pancreatic islet tumors upon inactivation of both Kmt2a and Men1. The Kmt2a/Men1 double knockout mouse model can be used as a mouse model to study advanced PanNETs.     

Novel engineered TRAIL-based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.     

Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer.     

Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

Background  

Pancreatic ductal adenocarcinoma shows a distinct apoptosis resistance, which contributes significantly to the aggressive nature of this tumor and constrains the effectiveness of new therapeutic strategies. Apoptosis resistance is determined by the net balance of the cells pro-and anti-apoptotic "control mechanisms". Numerous dysregulated anti-apoptotic genes have been identified in pancreatic cancer and seem to contribute to the high anti-apoptotic buffering capacity. We aimed to compare the benefit of simultaneous gene silencing (SGS) of several candidate genes with conventional gene silencing of single genes.     

p21 loss cooperates with INK4 inactivation facilitating immortalization and Bcl-2-mediated anchorage-independent growth of oncogene-transduced primary mouse fibroblasts

The INK4 and CIP cyclin-dependent kinase (Cdk) inhibitors (CKI) activate pocket protein function by suppressing Cdk4 and Cdk2, respectively. Although these inhibitors are lost in tumors, deletion of individual CKIs results in modest proliferation defects in murine models. We have evaluated cooperativity between loss of all INK4 family members (using cdk4r24c mutant alleles that confer resistant to INK4 inhibitors) and p21(Waf1/Cip1) in senescence and transformation of mouse embryo fibroblasts (MEF). We show that mutant cdk4r24c and p21 loss cooperate in pRb inactivation and MEF immortalization. Our studies suggest that cdk4r24c mediates resistance to p15(INK4B)/p16(INK4A) that accumulates over passage, whereas loss of p21 suppresses hyperoxia-induced Cdk2 inhibition and pRb dephosphorylation on MEF explantation in culture. Although cdk4r24c and p21 loss cooperate in H-ras(V12)/c-myc-induced foci formation, they are insufficient for oncogene-induced anchorage-independent growth. Interestingly, p21(-/-); cdk4r24c MEFs expressing H-ras(V12) and c-myc display detachment-induced apoptosis and are transformed by c-myc, H-ras(V12), and Bcl-2. We conclude that the INK4 family and p21 loss cooperate in promoting pRb inactivation, cell immortalization, and H-ras(V12)/c-myc-induced loss of contact inhibition. In addition, absence of pRb function renders H-ras(V12) + c-myc-transduced fibroblasts prone to apoptosis when deprived of the extracellular matrix, and oncogene-induced anchorage-independent growth of pocket protein-deficient cells requires apoptotic suppression.     

Targeting the Bcl-2 family in cancer therapy

The targeting of anti-apoptotic members of the Bcl-2 family of proteins, especially Bcl-2, is the focus of efforts to modulate the intrinsic pathway of apoptosis and effect tumor death. Current understanding of the role of Bcl-2 family proteins and strategies and recent developments in drug inhibitors of these proteins, particularly antisense oligonucleotides and targeted small molecules, are reviewed.     

Targeting sphingosine kinase 2 (SphK2) by ABC294640 inhibits colorectal cancer cell growth in vitro and in vivo

Background

Colorectal cancer (CRC) is a major health problem in China and around the world. It is one of the leading causes of cancer-related deaths. Research groups are thus searching for novel and more efficient anti-CRC agents.

Results

Here we demonstrated that ABC294640, a novel SphK2 inhibitor, induced growth inhibition and apoptosis in transformed and primary CRC cells. The SphK activity was remarkably inhibited by ABC294640, accompanied by sphingosine-1-phosphate (S1P) depletion and ceramide incensement in CRC cells. Exogenously-added S1P inhibited ABC294640-induced HT-29 cell lethality. While C6 ceramide and SphK1 inhibitor SKI-II facilitated ABC294640-induced cytotoxicity against HT-29 cells. ABC294640 inhibited AKT-S6K1, but activated JNK signaling in transformed and primary CRC cells. JNK inhibitors (SP600125 and JNKi-II) alleviated ABC294640-induced CRC cell apoptosis. Moreover, a low concentration of ABC294640 sensitized the activity of 5-FU and cisplatin in vitro. In vivo, ABC294640 oral administration dramatically inhibited HT-29 xenografts growth in nude mice.

Conclusions

Targeting of SphK2 by ABC294640 potently inhibits CRC cell growth both in vitro and in vivo, ABC294640 could be developed as a novel therapeutic for the treatment of CRC.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0205-y) contains supplementary material, which is available to authorized users.     

Loss of XIAP sensitizes rosiglitazone-induced growth inhibition of colon cancer in vivo

Ligands for peroxisome proliferator-activated receptor gamma (PPAR gamma) possess anticancer properties. However, the efficacy of PPAR gamma ligands varies in different cancers. In colon cancer, the role of PPAR gamma and its ligands is controversial. We recently showed that downregulation of X-linked inhibitor of apoptosis protein (XIAP) could sensitize colon cancer cells to troglitazone, and 15-deoxy-D12,14-prostaglandin J2 (15-PGJ2) induced cell killing. In our study, we aimed to examine whether rosiglitazone, another more clinically relevant PPAR gamma ligand, has any synergistic anticancer effect with XIAP downregulation in colon cancer. Human colon cancer cell lines HCT116-XIAP(+/+) cells and HCT116-XIAP(-/-) cells were treated with various concentrations of rosiglitazone. The effects of rosiglitazone on cell proliferation, apoptosis and growth of xenograft colon cancers were studied. Rosiglitazone barely suppressed the growth and only very weakly induced apoptosis in HCT116 cells in vitro. Loss of XIAP did not sensitize HCT116 cells to rosiglitazone-induced growth inhibition or apoptosis. In vivo studies revealed that rosiglitazone strongly suppressed the growth of xenograft colon cancer, especially tumors derived from HCT116-XIAP(-/-) cells. The rosiglitazone-treated tumor had reduced expression of ki-67 and lowered mitotic rate. Downregulation of XIAP was associated with an impaired activation of PPAR gamma by its ligand. Rosiglitazone induced marked upregulation of PTEN in HCT116-XIAP(-/-) cells, as well as in xenograft tumors derived from HCT116-XIAP(-/-) cells. We concluded that rosiglitazone significantly suppresses the growth of xenograft colon cancer, and downregulation of XIAP sensitizes the xenograft tumors to rosiglitazone-induced tumor suppression in vivo via upregulation of PTEN.     

靶向XIAP的siRNA抑制结肠癌LoVo细胞体内外的增殖

目的：研究siRNA干扰X连锁凋亡抑制蛋白（Xlinked inhibitor of apoptosis protein, XIAP）基因表达对结肠癌细胞LoVo体内外增殖的影响。方法：构建XIAP真核表达干扰载体pSil2.1shXIAP1和pSil2.1shXIAP2,脂质体转染结肠癌细胞株LoVo,G418筛选出稳定转染LoVoshXIAP2细胞。RTPCR和Western blotting方法检测XIAP mRNA和蛋白的表达。MTT法和平板克隆形成实验检测LoVo细胞的增殖;流式细胞仪检测细胞的凋亡;裸鼠移植瘤模型观察XIAP表达下调对结肠癌体内增殖活性的影响。结果：LoVoshXIAP2细胞中XIAP mRNA和蛋白表达水平均显著下调。相对于对照组细胞,LoVoshXIAP2细胞的增殖和克隆形成率显著降低(P<0.01）,凋亡率显著增加（P<0.01）。 LoVoshXIAP2移植瘤组织中XIAP蛋白表达明显下调,LoVoshXIAP2移植瘤生长显著抑制（均P<0.05）。结论：pSil2.1shXIAP2能够抑制结肠癌LoVo细胞中XIAP蛋白的表达,抑制LoVo细胞体内外的增殖,有望成为结肠癌免疫治疗的新手段。     

Overexpression of Bcl-2-associated death inhibits A549 cell growth in vitro and in vivo

The importance of apoptosis during the process of inhibiting tumorigenesis has been recognized. The role of BH3-only proapoptotic protein Bcl-2-associated death (BAD) in tumor growth remains controversial. The aim of this study was to explore the role of BAD in lung cancer cells. Our study showed that expression of BAD was upregulated in A549 cells by a recombinant lentivirus overexpressing BAD. In vitro, BAD overexpression significantly inhibited A549 cell proliferation and induced apoptosis in cell proliferation and apoptosis assays, respectively. The effect of BAD on A549 cells was studied in tumor xenograft of nude mice and the results showed that the tumor volume in the experimental group was smaller than the control groups. Further, immunohistochemical technique was used to determine the cell proliferation and apoptosis status of the lung tumor xenograft cells. This demonstrated that the in vivo and in vitro results were consistent. Taken together, our results indicate that overexpression of BAD inhibits the growth of A549 cells in vitro and in vivo, through inhibiting cell proliferation and inducing apoptosis. Thus, BAD could be a potential therapeutic target.     

Stable XIAP knockdown clones of HCT116 colon cancer cells are more sensitive to TRAIL, taxanes and irradiation in vitro

Purpose  To develop a model of X-linked inhibitor of apoptosis (XIAP) down regulation in colorectal cancer cell lines. This may be used to determine whether combination strategies have clinical potential. Methods  A series of clones were developed using short hairpin RNA (shRNA) against XIAP stably expressed in HCT116 cells. XIAP mRNA and protein levels were established by RT-PCR and Immunoblot, respectively. GeneChip microarrays confirmed XIAP knockdown and absence of compensation by other IAP members. Results  Four XIAP knockdown cell lines show 82–93% reduction in XIAP mRNA and 67–89% reduction in protein when compared to four luciferase control cell lines. XIAP knockdown sensitises cells to rhTRAIL by a factor of 3, to paclitaxel and docetaxel by a factor of >2 and, to a lesser extent, radiotherapy (20% enhancement). Conclusions  Clinical trials with XIAP antisense continue, and these data suggest combination studies with agents such as rhTRAIL and taxanes should be undertaken.     

Targeting mTORC2 inhibits colon cancer cell proliferation in vitro and tumor formation in vivo

The mammalian target of rapamycin (mTOR), which exists in two functionally distinct complexes, mTORC1 and mTORC2 plays an important role in tumor growth. Whereas the role of mTORC1 has been well characterized in this process, little is known about the functions of mTORC2 in cancer progression. In this study, we explored the specific role of mTORC2 in colon cancer using a short hairpin RNA expression system to silence the mTORC2-associated protein rictor. We found that downregulation of rictor in HT29 and LS174T colon cancer cells significantly reduced cell proliferation. Knockdown of rictor also resulted in a G1 arrest as observed by cell cycle analysis. We further observed that LS174T cells deficient for rictor failed to form tumors in a nude mice xenograft model. Taken together, these results show that the inhibition of mTORC2 reduces colon cancer cell proliferation in vitro and tumor xenograft formation in vivo. They also suggest that specifically targeting mTORC2 may provide a novel treatment strategy for colorectal cancer.     

Targeting Bcl-2 family proteins in adult T-cell leukemia/lymphoma: in vitro and in vivo effects of the novel Bcl-2 family inhibitor ABT-737

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-1). ABT-737, a small molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, significantly induced apoptosis in HTLV-1 infected T-cell lines as well as in fresh ATLL cells, and synergistically enhanced the cytotoxicity and apoptosis induced by conventional cytotoxic drugs. Moreover, ABT-737 significantly inhibited the in vivo tumor growth of an ATLL mouse model. These results suggest that the use of an agent targeting anti-apoptotic bcl-2 family proteins, either alone or in combination with other conventional drugs, represents a novel promising approach for ATLL.     

Src and ADAM-17-mediated shedding of transforming growth factor-alpha is a mechanism of acute resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL.     

Ribozyme targeting of HER-2 inhibits pancreatic cancer cell growth in vivo

We have analysed HER-2 expression and function in pancreatic cancer cells to determine whether HER-2 has a rate-limiting role for pancreatic cancer cell growth in vitro and in vivo. To specifically assess HER-2 function, we used HER-2-targeted ribozymes expressed under the control of the tet-off promoter system. Six out of 11 human pancreatic cancer cell lines expressed all four epidermal growth factor (EGF)-receptor family members (HER-1 (EGF-R), HER-2, HER-3, and HER-4), including Panc89 cells. Expression of the ribozymes quenched endogenous HER-2 mRNA levels in Panc89 cells by approximately 40-60% which was reflected by a 40-50% reduction of the HER-2 surface glycoprotein. HER-2 depletion inhibited the in vitro proliferation rate by approximately 40% and decreased in vivo tumour growth by approximately 60% (P<0.05). Our study demonstrates for the first time a rate-limiting role for HER-2 in pancreatic cancer cell proliferation and suggests HER-2 targeting as a potential approach in pancreatic cancer therapy.     

Chetomin induces degradation of XIAP and enhances TRAIL sensitivity in urogenital cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising anti-cancer agents, but some tumor types develop resistance to TRAIL. Here, we report that chetomin, an inhibitor of hypoxia-inducible factors, is a potent enhancer of TRAIL-induced apoptosis. TRAIL or chetomin alone weakly induced apoptosis, but the combination of chetomin and TRAIL synergistically induced apoptosis in prostate cancer PC-3 cells. The combination of chetomin and TRAIL induces the activation of caspase-3, -8, -9 and -10. Among the apoptotic factors related to the TRAIL pathway, chetomin markedly decreased the X-linked inhibitor of apoptosis (XIAP) protein levels in a dose-dependent manner, but other IAP family members, TRAIL receptors and Bcl-2 family members were not altered by chetomin. Using XIAP siRNA instead of chetomin, down-regulation of XIAP sensitized PC-3 cells to TRAIL-induced apoptosis. Conversely, transient transfection of XIAP reduced the apoptotic response to combined treatment with chetomin and TRAIL. Treatment with chetomin induced a rapid decrease in XIAP protein levels but had no effect on XIAP mRNA levels. Since chetomin-mediated XIAP down-regulation was completely prevented by proteasome inhibitors, it was suggested that chetomin induces the degradation of the XIAP protein in a proteasome-dependent manner. Additionally, chetomin also sensitized renal cancer Caki-1 cells and bladder cancer UM-UC-3 cells to TRAIL-induced apoptosis via down-regulation of XIAP. Co-treatment of chetomin and TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMC). Taken together, these findings suggest that TRAIL and chetomin synergistically induce apoptosis in human urogenital cancer cells through a mechanism that involves XIAP down-regulation by chetomin.