Targeting CD24 for treatment of ovarian cancer by short hairpin RNA

Background aimsCD24 is markedly overexpressed in ovarian cancer and plays a critical role in ovarian cancer survival and metastasis, rendering it an interesting target for anti-tumor therapy. Using short hairpin RNA (shRNA) targeting CD24, we aimed to investigate the anti-tumor efficacy of CD24 knockdown in ovarian cancer cells in vitro and in vivo.MethodsCD24 shRNA vector (CD24–shRNA) and empty plasmid vector (EP) were transfected into ovarian cancer SKOV3 cells and the knockdown efficacy assessed by Western blot analysis. The effects of CD24 knockdown in SKOV3 cells in vitro, including cell viability and apoptosis, were determined using methyl thiazolyl blue tetrazolium bromide (MTT), flow cytometry and propidium iodide (PI) staining assays. The effects in vivo of CD24 knockdown on angiogenesis, cell proliferation and apoptosis were assessed using immunohistochemistry against CD31, proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assays.ResultsTransfection of CD24–shRNA effectively down-regulated CD24 expression in vitro and in vivo. Administration of CD24–shRNA into nude mice bearing ovarian cancer significantly suppressed tumor volume growth.ConclusionsKnockdown of CD24 expression by CD24–shRNA significantly inhibited cell viability and induced apoptosis of SKOV3 cells in vitro. Administration with CD24–shRNA in vivo suppressed tumor volume increase by microvessel density (MVD) decrease, cell proliferation inhibition and apoptosis induction. All the data suggested that knockdown of CD24 by shRNA might be a potential therapeutic approach against human ovarian cancer.     

Anti-Cancer Activity of an Osthole Derivative,NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.     

siRNA‐induced CD44 knockdown suppresses the proliferation and invasion of colorectal cancer stem cells through inhibiting epithelial–mesenchymal transition

CD44 has shown prognostic values and promising therapeutic potential in multiple human cancers; however, the effects of CD44 silencing on biological behaviors of cancer stem cells (CSCs) have not been fully understood in colorectal cancer. To examine the contribution of siRNA‐induced knockdown of CD44 to the biological features of colorectal CSCs, colorectal CSCs HCT116‐CSCs were generated, and CD44 was knocked down in HCT116‐CSCs using siRNA. The proliferation, migration and invasion of HCT116‐CSCs were measured, and apoptosis and cell‐cycle analyses were performed. The sensitivity of HCT116‐CSCs to oxaliplatin was tested, and xenograft tumor growth assay was performed to examine the role of CD44 in HCT116‐CSCs tumorigenesis in vivo. In addition, the expression of epithelial–mesenchymal transition (EMT) markers E‐cadherin, N‐cadherin and vimentin was quantified. siRNA‐induced knockdown of CD44 was found to inhibit the proliferation, migration and invasion, induce apoptosis, promote cell‐cycle arrest at the G1/G0 phase and increase the sensitivity of HCT116‐CSCs to oxaliplatin in HCT116‐CSCs, and knockdown of CD44 suppressed in vivo tumorigenesis and intrapulmonary metastasis of HCT116‐CSCs. Moreover, silencing CD44 resulted in EMT inhibition. Our findings demonstrate that siRNA‐induced CD44 knockdown suppresses the proliferation, invasion and in vivo tumorigenesis and metastasis of colorectal CSCs by inhibiting EMT.     

Overexpression of the Promigratory and Prometastatic PTK7 Receptor Is Associated with an Adverse Clinical Outcome in Colorectal Cancer

Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.     

Methylselenol,a selenium metabolite,modulates p53 pathway and inhibits the growth of colon cancer xenografts in Balb/c mice

It is has been hypothesized that methylselenol is a critical selenium metabolite for anticancer activity in vivo. In this study, we used a protein array which contained 112 different antibodies known to be involved in the p53 pathway to investigate the molecular targets of methylselenol in human HCT116 colon cancer cells. The array analysis indicated that methylselenol exposure changed the expression of 11 protein targets related to the regulation of cell cycle and apoptosis. Subsequently, we confirmed these proteins with the Western blotting approach, and found that methylselenol increased the expression of GADD 153 and p21 but reduced the level of c-Myc, E2F1 and Phos p38 MAP kinase. Similar to our previous report on human HCT116 colon cancer cells, methylselenol also inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase in mouse colon cancer MC26 cells. When the MC26 cells were transplanted to their immune-competent Balb/c mice, methylselenol-treated MC26 cells had significantly less tumor growth potential than that of untreated MC26 cells. Taken together, our data suggest that methylselenol modulates the expression of key genes related to cell cycle and apoptosis and inhibits colon cancer cell proliferation and tumor growth.     

Lentivirus-mediated siRNA targeting SAE1 induces cell cycle arrest and apoptosis in colon cancer cell RKO

Deregulated expression of proteins involved in the SUMOylation pathway has been detected in several tumors. SUMO1-activating enzyme subunit 1 (SAE1) plays an important role in this process. We found that SAE1 was highly expressed in the colon cancer cell line RKO and used lentivirus-mediated siRNA to suppress SAE1 expression in RKO cells. RNA-interference efficiently and specifically downregulated the target gene expression in RKO on both mRNA and protein levels. Silencing of SAE1 inhibited proliferation and reduced colony formation of RKO cells. Furthermore, as it has been shown by flow cytometry analysis, specific knockdown of SAE1 slowed down the cell population at G0/G1 phase and induced apoptosis of RKO cells. On the base of results obtained, one can suppose the biological significance of SAE1 in colon tumorigenesis and a use of this protein as a novel molecular target for colon cancer therapy.     

Valosin-containing protein (VCP) promotes the growth,invasion, and metastasis of colorectal cancer through activation of STAT3 signaling

Valosin-containing protein (VCP) was previously shown to exhibit high expression in colorectal cancer (CRC) tissues as compared with that in normal tissues; however, the role of VCP in human CRC cells has remained to be elucidated. Two colorectal cancer cell lines HCT116 and RKO were used in the experiment. We introduced lentiviral constructs expressing VCP to infect RKO cells and lenti-shRNA targeting VCP into HCT116 cells, respectively. Cell proliferation, invasion, apoptosis, and cell cycle arrest were subsequently examined by MTT assay, transwell chamber assay, flow cytometry, and western blot analysis, respectively. Furthermore, a subcutaneous tumor mouse model and lung metastasis model was used to investigate the effects of VCP on the growth and metastasis of CRC cells in vivo. VCP knockdown was shown to inhibit cell proliferation, chemoresistance and invasion, and induce apoptosis in the HCT116 CRC cells, whereas VCP over-expression suppressed apoptosis and chemoresponse, promoted proliferation and invasion of the RKO CRC cells. In addition, in the subcutaneous tumor and lung metastasis mouse model, VCP knockdown in HCT116 cells suppressed carcinogenesis and metastasis in vivo. The findings of the present study indicated that VCP is very important for the proliferation and metastasis of CRC; therefore, targeting VCP and its downstream targets may represent novel therapies for the treatment of CRC.

     

CtIP promotes G2/M arrest in etoposide-treated HCT116 cells in a p53-independent manner

Acquired resistance to cytotoxic antineoplastic agents is a major clinical challenge in tumor therapy; however, the mechanisms involved are still poorly understood. In this study, we show that knockdown of CtIP, a corepressor of CtBP, promotes cell proliferation and alleviates G2/M phase arrest in etoposide (Eto)-treated HCT116 cells. Although the expression of p21 and growth arrest and DNA damage inducible α (GADD45a), which are important targets of p53, was downregulated in CtIP-deficient HCT116 cells, p53 deletion did not affect G2/M arrest after Eto treatment. In addition, the phosphorylation levels of Ser317 and Ser345 in Chk1 and of Ser216 in CDC25C were lower in CtIP-deficient HCT116 cells than in control cells after Eto treatment. Our results indicate that CtIP may enhance cell sensitivity to Eto by promoting G2/M phase arrest, mainly through the ATR-Chk1-CDC25C pathway rather than the p53-p21/GADD45a pathway. The expression of CtIP may be a useful biomarker for predicting the drug sensitivity of colorectal cancer cells.     

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.     

Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.     

Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment

Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term “clonal dominance”. We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780^cis) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.     

Knockdown of pyruvate kinase type M2 suppresses tumor survival and invasion in osteosarcoma cells both in vitro and in vivo

Osteosarcoma (OS) is the mostly diagnosed primary bone malignancy. Emerging evidence indicates that the activity of pyruvate kinase M2 (PKM2) isoform is crucial for the survival of tumor cells. In the present study, the effect of PKM2 knockdown on the proliferation and migration of OS cells were assessed both in vitro and in vivo. Small hairpin RNA (shRNA) technology were employed to suppress the expression of PKM2 in MG-63 and Saos-2 cell lines. In vitro, shRNA-mediated knockdown of PKM2 efficiently inhibited cell proliferation, and induced G1 cell cycle arrest and apoptosis in both cell lines, which was associated with decreased expressions of cyclin D1 and Bcl-2 as well as increased expressions of Bax, cleaved-caspase-3, and cleaved-PARP. The invasion and migration potential of OS cell lines were also inhibited by PKM2 knockdown through the regulating effect of PKM2 on MMP-2 and VEGF signaling. In vivo, knockdown of PKM2 decelerated tumor growth rate and induced structure deterioration in tumor tissues. The current study for the first time showed that the activity of PKM2 was indispensable for the development and metastasis of OS, thereby providing the basic information for the future development of PKM2-based anti-OS therapies.     

Silencing of Jagged1 inhibits cell growth and invasion in colorectal cancer

Dysregulated Notch signaling has a critical role in the tumorigenesis. Jagged1, a Notch ligand, is overexpressed in various human cancers. Recent studies revealed the involvement of Jagged1 in colorectal cancer (CRC) development. These basic studies provide a promising potential for inhibition of the Notch pathway for the treatment of CRC. Herein, we aimed to investigate the consequences of targeting Jagged1 using shRNA on CRC both in vitro and in vivo to test their potential to inhibit this key element for CRC treatment. We found that downregulation of Jagged1 with lentiviral Jagged1-shRNA resulted in decreased colon cancer cell viability in vitro, most likely mediated through reduced cell proliferation. Importantly, Jagged1 knockdown induced G₀/G₁ phase cell cycle arrest, with reduced Cyclin D1, Cyclin E and c-Myc expression. Silencing of Jagged1 reduced the migration and invasive capacity of the colon cancer cells in vitro. Furthermore, colon cancer cells with knockdown of Jagged1 had much slower growth rate than control cells in a xenograft mouse model in vivo, with a marked downregulation of cell proliferation markers (PCNA, Ki-67, and c-Myc) and metastasis markers (MMP-2 and MMP-9). These findings rationalize a mechanistic approach to CRC treatment based on Jagged1-targeted therapeutic development.     

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

Background

Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).

Materials and Methods

HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.

Results

Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.

Conclusions

We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.     

Apoptin induces pyroptosis of colorectal cancer cells via the GSDME-dependent pathway

Apoptin is a small molecular weight protein encoded by the VP3 gene of chicken anemia virus (CAV). It can induce apoptosis of tumor cells and play anti-tumorigenic functions. In this study, we identified a time-dependent inhibitory role of apoptin on the viability of HCT116 cells. We also demonstrated that apoptin induces pyroptosis through cleaved caspase 3, and with a concomitant cleavage of gasdermin E (GSDME) rather than GSDMD. GSDME knockdown switched the apoptin-induced cell death from pyroptosis to apoptosis in vitro. Furthermore, we demonstrated that the effect of apoptin on GSDME-dependent pyroptosis could be mitigated by caspase-3 and caspase-9 siRNA knockdown. Additionally, apoptin enhanced the intracellular reactive oxygen species (ROS), causing aggregation of the mitochondrial membrane protein Tom20. Moreover, bax and cytochrome c were released to the activating caspase-9, eventually triggering pyroptosis. Therefore, GSDME mediates the apoptin-induced pyroptosis through the mitochondrial apoptotic pathway. Finally, using nude mice xenografted with HCT116 cells, we found that apoptin induces pyroptosis and significantly inhibits tumor growth. Based on this mechanism, apoptin may provide a new strategy for colorectal cancer therapy.     

SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.