Kangjian Zhang

A new oncolytic adenoviral vector carrying dual tumour suppressor genes shows potent anti-tumour effect

Cancer Targeting Gene‐Viro‐Therapy (CTGVT) is a promising cancer therapeutical strategy that strengthens the anti‐tumour effect of oncolytic virus by expressing inserted foreign anti‐tumour genes. In this work, we constructed a novel adenoviral vector controlled by the tumour‐specific survivin promoter on the basis of the ZD55 vector, which is an E1B55KD gene deleted vector we previously constructed. Compared with the original ZD55 vector, this new adenoviral vector (ZD55SP/E1A) showed much better ability of replication and reporter gene expression. We then combined anti‐tumour gene interleukine‐24 (IL‐24) with an RNA polymerase III‐dependent U6 promoter driving short hairpin RNA (shRNA) that targets M‐phase phosphoprotein 1 (MPHOSPH1, a newly identified oncogene) by inserting the IL‐24 and the shRNA of MPHOSPH1 (shMPP1) expression cassettes into the new ZD55SP/E1A vector. Our results demonstrated excellent anti‐tumour effect of ZD55SP/E1A‐IL‐24‐shMPP1 in vitro on multiple cancer cell lines such as lung cancer, liver cancer and ovarian caner. At high multiplicity‐of‐infection (MOI), ZD55SP/E1A‐IL‐24‐shMPP1 triggered post‐mitotic apoptosis in cancer cells by inducing prolonged mitotic arrest; while at low MOI, senescence was induced. More importantly, ZD55SP/E1A‐IL‐24‐shMPP1 also showed excellent anti‐tumour effects in vivo on SW620 xenograft nude mice. In conclusion, our strategy of constructing an IL‐24 and shMPP1 dual gene expressing oncolytic adenoviral vector, which is regulated by the survivin promoter and E1B55KD deletion, could be a promising method of cancer gene therapy.

Complete Eradication of Xenograft Hepatoma by Oncolytic Adenovirus ZD55 Harboring TRAIL-IETD-Smac Gene with Broad Antitumor Effect

Cancer-targeting dual-gene virotherapy (CTGVT-DG) is an important modification of CTGVT, in which two suitable genes are used to obtain an excellent antitumor effect. A key problem is to join the two genes to form one fused gene, and then to clone it into the oncolytic viral vector so that only one investigational new drug application, instead of two, is required for clinical use. Many linkers (e.g., internal ribosome entry site) are used to join two genes together, but they are not all equally efficacious. Here, we describe finding the best linker, that is, sequence encoding the four amino acids IETD, to join the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene and the second mitochondria-derived activator of caspase (Smac) gene to form TRAIL-IETD-Smac and inserting it into oncolytic viral vector ZD55 to construct ZD55-TRAIL-IETD-Smac, which matched ZD55-TRAIL plus ZD55-Smac in completely eliminating xenograft hepatoma. ZD55-TRAIL-IETD-Smac works by quantitative cleavage at IETD↓by inducing caspase-8; activation or inhibition of caspase-8 could up- or downregulate cleavage, respectively. The cleaved product, TRAIL-IETD, does not affect the function of TRAIL. Numerous experiments have shown that the combined use of ZD55-TRAIL plus ZD55-X could completely eradicate many xenograft tumors, and therefore the IETD is potentially a useful linker to construct many antitumor drugs, for example, ZD55-TRAIL-IETD-X, where X has a compensative or synergetic effect on TRAIL. We found that the antitumor effect of ZD55-IL-24-IETD-TRAIL also has an equivalent antitumor effect compared with the combined use of ZD55-IL-24 plus ZD55-TRAIL, because ZD55-IL-24 could also induce caspase-8. This means that IETD, as a two-gene linker, may have broad use.

SNORD126 promotes HCC and CRC cell growth by activating the PI3K-AKT pathway through FGFR2

Small nucleolar RNA (snoRNA) dysfunctions have been associated with cancer development. SNORD126 is an orphan C/D box snoRNA that is encoded within introns 5-6 of its host gene, cyclin B1-interacting protein 1 (CCNB1IP1). The cancer-associated molecular mechanisms triggered by SNORD126 are not fully understood. Here, we demonstrate that SNORD126 is highly expressed in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) patient samples. SNORD126 increased Huh-7 and SW480 cell growth and tumorigenicity in nude mice. Knockdown of SNORD126 inhibited HepG2 and LS174T cell growth. We verified that SNORD126 was not processed into small RNAs with miRNA activity. Moreover, SNORD126 did not show a significant expression correlation with CCNB1IP1 in HCC samples or regulate CCNB1IP1 expression. Our gene expression profile analysis indicated that SNORD126-upregulated genes frequently mapped to the PI3K-AKT pathway. SNORD126 overexpression increased the levels of phosphorylated AKT, GSK-3β, and p70S6K and elevated fibroblast growth factor receptor 2 (FGFR2) expression. siRNA-mediated knockdown or AZD4547-mediated inactivation of FGFR2 in SNORD126-overexpressing Huh-7 cells inhibited AKT phosphorylation and suppressed cell growth. These findings indicate an oncogenic role for SNORD126 in cancer and suggest its potential as a therapeutic target.

Potent and Specific Antitumor Effect for Colorectal Cancer by CEA and Rb Double Regulated Oncolytic Adenovirus Harboring ST13 Gene

Cancer Targeting Gene-Viro-Therapy (CTGVT) is constructed by inserting an antitumor gene into an oncolytic virus (OV). It is actually an OV-gene therapy, which has much better antitumor effect than either gene therapy alone or virotherapy alone in our previously published papers. This study is a modification of CTGVT by inserting a colorectal cancer (CRC) specific suppressor gene, ST13, into a CRC specific oncolytic virus, the Ad·CEA·E1A(Δ24), to construct the Ad·(ST13)·CEA·E1A(Δ24) for increasing the targeting tropism to colorectal cancer and it was briefly named as CTGVT-CRC. Although many studies on CEA promoter and ST13 gene were reported but no construct has been performed to combine both of them as a new strategy for colorectal cancer (CRC) specific therapy. In addition to the CRC specificity, the antitumor effect of Ad·(ST13)·CEA·E1A(Δ24) was also excellent and got nearly complete inhibition (not eradication) of CRC xenograft since ST13 was an effective antitumor gene with less toxicity, and a Chinese patent (No. 201110319434.4) was available for this study. Ad·(ST13)·CEA·E1A(Δ24) caused cell apoptosis through P38 MAPK (i.e. P38) which upregulated CHOP and ATF2 expression. The mitochondrial medicated apoptosis pathway was activated by the increase of caspase 9 and caspase 3 expression.

Synergistic antitumor effect of TRAIL and IL-24 with complete eradication of hepatoma in the CTGVT-DG strategy.

The ZD55-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ZD55-interleukin (IL)-24 were constructed by inserting TRAIL or IL-24 gene separately into the oncolytic adenovirus named ZD55 (with adenovirus E1B-55kD deletion). The resulting ZD55-TRAIL and ZD55-IL-24 were used in combination to treat xenograft tumors in nude mice model. The results showed that it can not only completely eliminate BEL7404 hepatoma xenograft but also have excellent antitumor effect against gaster, lung, prostate, and breast carcinomas. It was also found that ZD55-TRAIL could not only suppress the tumor growth promoting effect by ZD55-IL-24 at lower dosage, but also substantially reduce the cancer cell viability in their combined use. This is because ZD55-IL-24 and ZD55-TRAIL could mutually enhance each others antitumor effect greatly. All these findings conspicuously showed the synergistic antitumor effect of TRAIL and IL-24, which is also the reason for the antitumor effect by the combined use of TRAIL and IL-24 in vitro and also in vivo.

Interferon-related secretome from direct interaction between immune cells and tumor cells is required for upregulation of PD-L1 in tumor cells

PD-L1, also known as CD274, plays a vital role in tumor cell related immune escape. It can be expressed on the cell surface of many solid tumors (Brahmer et al., 2012) and inhibits T cell proliferation and cytokine production by binding to the Tcell surface receptor programmed death 1 (PD-1) or B7-1 (McClanahan et al., 2015). In 2013, targeting PD-1/ PD-L1 signaling for cancer immunotherapy was selected as the No.1 scientific breakthrough of the year by the editors of Science. Interferons (IFNs) are a group of pleiotropic cytokines, demonstrated anti-viral, anti-tumor, and immune regulatory functions (York et al., 2015). Type I interferon binds a heterodimeric receptor composed of IFNAR1 and IFNAR2. This activates a canonical JAK/STAT signaling pathway that ultimately induces a set of interferon-stimulated genes to exert its biological activity (Ejlerskov et al., 2015). Recently, PD-L1 was reported to be downstream of IFN signaling in human oral squamous carcinoma, melanoma, and human acute myeloid leukemia blast cells (Chen et al., 2012; Furuta et al., 2014; Kronig et al., 2014). The tumor microenvironment plays an important role in tumor growth and metastasis. Different components of the tumor microenvironment such as T cells, B cells, NK cells, dendritic cells, mast cells, granulocytes, Treg cells, myeloid derived suppressor cells (MDSC), and tumor associated macrophages (TAM) are recruited by different pathways (Joyce and Fearon, 2015). Tumor cells have been shown to upregulate PD-L1 after interacting with infiltrating immune cells (Cho et al., 2011; Hou et al., 2014), but the mechanism by which this occurs is not well understood. In this study, we found that PD-L1 upregulation in tumors was dependent on direct interaction with immune cells and was driven by a secreted factor such as type I interferon after cell-cell contact. Previous studies have demonstrated a positive correlation between tumor-infiltrating immune cells and elevated PD-L1 expression in tumor cells, but the mechanism by which this occurs is poorly understood. To investigate this, we co-cultured murine B16F10 melanoma cells with syngeneic splenocytes for 48 h. In addition, to determine whether direct cell contact is required for immune cell-mediated PD-L1 expression, the two types of cells were separated by a transwell-membrane that blocked their direct cell-cell interactions. Furthermore, another condition was tested in which B16F10 cells and immune cells were co-cultured in the plate and B16F10 cells were cultured in the transwell insert (Fig. 1A). Then the non-adherent immune cells were removed and B16F10 cells were harvested and analyzed for PD-L1 expression by flow cytometry. PD-L1 was more highly expressed in B16F10 cells that were co-cultured with splenocytes than in those cultured alone (Fig. 1B). However, PD-L1 expression was not increased in B16F10 cells separated from the splenocytes by a transwell membrane. We also found that a B16F10-splenocyte co-culture was able to induce PD-L1 in tumor cells separated from the co-culture by a transwell membrane (Fig. 1B). These effects were also observed in PD-L1 mRNA level changes by qPCR (Fig. 1C). These results suggested that active factors were secreted into the supernatant after the direct cell-cell interaction that was able to induce PD-L1 expression in tumor cells. To identify whether the regulation of PD-L1 was indeed driven by a secreted factor, B16F10 cells and splenocytes were co-cultured for 48 h. The supernatant was collected and centrifuged, and then used to treat B16F10 cells independently. The corresponding supernatant derived from B16F10 cells and splenocytes alone was also used to treat B16F10 cells as control groups (Fig. 1D). After 24 h, B16F10 cells treated with supernatant from the co-culture expressed more PD-L1 than cells treated with supernatant from the control mono-cultures (Fig. 1E and 1F). In addition, co-cultures of B16F10 cells with bone marrow (BM)-derived cells (Fig. 1G) or lymph node (LN)-derived cells also upregulated PD-L1 expression (Fig. 1H). To determine whether a similar effect would be seen in other types of cancer cells, additional studies on MC38 and Hepa1-6 cells

RGD-modified oncolytic adenovirus-harboring shPKM2 exhibits a potent cytotoxic effect in pancreatic cancer via autophagy inhibition and apoptosis promotion

The M2 isoform of pyruvate kinase (PKM2) is a key driver of glycolysis in cancer cells and has critical ‘non-metabolic’ functions in some cancers; however, the role of PKM2 in pancreatic cancer remains unclear. The aim of the current study was to elucidate the role of PKM2 in pancreatic cancer progression and the potential of PKM2 as a therapeutic target. In this study, we observed that PKM2 is highly expressed in patients with pancreatic cancer and is correlated to survival. Elevated PKM2 expression promoted cell proliferation, migration and tumor formation. The inhibition of cell growth by silencing PKM2 is caused by impairment of the autophagy process. To test the potential effects of downregulating PKM2 as a clinical therapy, we constructed an RGD-modified oncolytic adenovirus containing shPKM2 (OAd.R.shPKM2) to knock down PKM2 in pancreatic cancer cells. Cells transduced with OAd.R.shPKM2 exhibited decreased cell viability, and, in a PANC-1 xenograft model, intratumoral injection of OAd.R.shPKM2 resulted in reduced tumor growth. Furthermore, OAd.R.shPKM2 induced apoptosis and impaired autophagy in PANC-1 cells. Our results suggested that targeting PKM2 with an oncolytic adenovirus produced a strong antitumor effect, and that this strategy could broaden the therapeutic options for treating pancreatic cancer.

Possibility to Partly Win the War Against Cancer

We have worked out two strong antitumor strategies, namely, two antitumor stars. One is the “Cancer Targeting Gene-Viro-Therapy, CTGVT” strategy and the other is the super interferon (sIFN-I) protein. By the combination of the above two antitumor stars with other biotherapy such as antibodies, CIK (which will be described below) and surgery, chemotherapy, and radiotherapy, we can partly win the war against cancer.