Augmented antitumor activity of 5‐fluorouracil by double knockdown of MDM4 and MDM2 in colon and gastric cancer cells

Inactivation of the TP53 tumor suppressor gene is essential during cancer development and progression. Mutations of TP53 are often missense and occur in various human cancers. In some fraction of wild‐type (wt) TP53 tumors, p53 is inactivated by upregulated murine double minute homolog 2 (MDM2) and MDM4. We previously reported that simultaneous knockdown of MDM4 and MDM2 using synthetic DNA‐modified siRNAs revived p53 activity and synergistically inhibited in vitro cell growth in cancer cells with wt TP53 and high MDM4 expression (wtTP53/highMDM4). In the present study, MDM4/MDM2 double knockdown with the siRNAs enhanced 5‐fluorouracil (5‐FU)‐induced p53 activation, arrested the cell cycle at G₁ phase, and potentiated the antitumor effect of 5‐FU in wtTP53/highMDM4 human colon (HCT116 and LoVo) and gastric (SNU‐1 and NUGC‐4) cancer cells. Exposure to 5‐FU alone induced MDM2 as well as p21 and PUMA by p53 activation. As p53‐MDM2 forms a negative feedback loop, enhancement of the antitumor effect of 5‐FU by MDM4/MDM2 double knockdown could be attributed to blocking of the feedback mechanism in addition to direct suppression of these p53 antagonists. Intratumor injection of the MDM4/MDM2 siRNAs suppressed in vivo tumor growth and boosted the antitumor effect of 5‐FU in an athymic mouse xenograft model using HCT116 cells. These results suggest that a combination of MDM4/MDM2 knockdown and conventional cytotoxic drugs could be a promising treatment strategy for wtTP53/highMDM4 gastrointestinal cancers.     

TLR3 induction by anticancer drugs potentiates poly I:C‐induced tumor cell apoptosis

Toll‐like receptor 3 (TLR3) has gained recognition as a novel molecular target for cancer therapy because TLR3 activation by its synthetic ligand poly I:C directly causes tumor cell death. Recently, we reported that tumor suppressor p53 increases the expression of TLR3 in several tumor cell lines. Another study also showed that interferon‐α (IFN‐α) up‐regulates TLR3 expression. We thus hypothesized that various anticancer drugs such as p53‐activating reagents and IFNs may potentiate poly I:C‐induced tumor cell death through the up‐regulation of TLR3 expression. Here, we screened several anticancer drugs that, together with poly I:C, effectively cause tumor cell death in colon carcinoma HCT116 cells. We found that the DNA‐damaging reagent 5‐fluorouracil (5‐FU) increased TLR3 mRNA expression and potentiated poly I:C‐induced apoptosis in HCT116 p53^+/+ cells but had only minimal effect in p53^?/? cells, indicating a p53‐dependent pathway. On the other hand, IFN‐α increased poly I:C‐induced apoptosis and the TLR3 mRNA level in HCT116 p53^+/+ and p53^?/? cell lines. Furthermore, the combination of poly I:C, 5‐FU and IFN‐α induced the highest apoptosis in HCT116 p53^+/+ and p53^?/? cells. Taken together, these data suggest that the anticancer drugs increased TLR3 expression and subsequently potentiated poly I:C‐induced apoptosis likely via p53‐dependent and ‐independent pathways. Considering that the p53 status in malignant cells is heterogeneous, this combination approach may provide a highly effective tumor therapy. (Cancer Sci 2010)     

A phosphomimetic mutant of RelA/p65 at Ser536 induces apoptosis and senescence: An implication for tumor‐suppressive role of Ser536 phosphorylation

Hundreds of NF‐κB inhibitors have been developed for cancer therapy, but their clinical efficacy is unsatisfactory. Here we show that the phosphorylation activation at Ser536 of RelA/p65 protein, a main subunit in the NF‐κB family, may play a tumor‐suppressive role. In normal colon mucosa, RelA/p65 phosphorylation at Ser536 was increasingly increased with the maturation and apoptotic shedding of epithelial cells, but the phosphorylation at Ser536 was decreased in colon cancer. In colon (HCT116 p53 wt and p53 ?/?), breast (MCF7), and prostate (LNCaP and DU145) cancer cells, a phosphomimetic mutation of RelA/p65 at Ser536 (named p65/S536D) triggered dramatic apoptosis through affecting expression of a wide range of cell death/survival genes, such as Bim, Puma, Noxa, Bcl‐2 and survivin. In HCT116 cells, p65/S536D mutant upregulated Fas, insulted mitochondrial membrane potential, and triggered cleavage and activation of caspase‐3, 7, 8 and 9. A FasL neutralizing antibody (NOK1) prevented cell death induced by the p65/S536D. A pan inhibitor of caspases, Z‐VAD‐FMK (20 μM), blocked caspase‐mediated mitochondrial membrane depolarization. This p65/S536D also triggered senescence in HCT116 cells through a p16‐dependent pathway, but not in MFC7 due to lack of p16. Intratumoral delivery of the p65/S536D effectively suppressed tumor growth in nude mice. Together our data suggest that the phosphorylation of RelA/p65 at Ser536 may confer it a tumor‐suppressive role by inducing apoptosis and senescence, highlighting the importance of discriminating the function and active status of individual active sites in RelA/p65 when NF‐κB inhibitors are considered for targeted therapy of cancer.     

53BP1 suppresses epithelial–mesenchymal transition by downregulating ZEB1 through microRNA‐200b/429 in breast cancer

Epithelial–mesenchymal transition (EMT) is an important mechanism of cancer invasion and metastasis. Although p53 binding protein 1 (53BP1) has been implicated in several biological processes, its function in EMT of human cancers has not yet been reported. Here, we show that 53BP1 negatively regulated EMT by modulating ZEB1 through targeting microRNA (miR)‐200b and miR‐429. Furthermore, 53BP1 promoted ZEB1‐mediated upregulation of E‐cadherin and also inhibited the expressions of mesenchymal markers, leading to increased migration and invasion in MDA‐MB‐231 breast cancer cells. Consistently, in MCF‐7 breast cancer cells, low 53BP1 expression reduced E‐cadherin expression, resulting in increased migration and invasion. These effects were reversed by miR‐200b and miR‐429 inhibition or overexpression. Sections of tumor xenograft model showed increased ZEB1 expression and decreased E‐cadherin expression with the downregulation of 53BP1. In 18 clinical tissue samples, expression of 53BP1 was positively correlated with miR‐200b and mir‐429 and negatively correlated with ZEB1. It was also found that 53BP1 was associated with lymph node metastasis. Taken together, these results suggest that 53BP1 functioned as a tumor suppressor gene by its novel negative control of EMT through regulating the expression of miR‐200b/429 and their target gene ZEB1.     

MiRNA‐362‐3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan^® Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR‐362‐3p, miR‐570, miR‐148 a* and miR‐944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR‐362‐3p in a second independent cohort of 43 CRC patients, using single TaqMan^® microRNA assays. In vitro functional analysis showed that over‐expression of miR‐362‐3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR‐362‐3p targets by mRNA profiling of HCT116 cells over‐expressing miR‐362‐3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR‐362‐3p over‐expression. We conclude that miR‐362‐3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR‐362‐3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.     

Regulation of ITGA3 by the anti‐tumor miR‐199 family inhibits cancer cell migration and invasion in head and neck cancer

For patients with head and neck squamous cell carcinoma (HNSCC), survival rates have not improved due to local recurrence and distant metastasis. Current targeted molecular therapies do not substantially benefit HNSCC patients. Therefore, it is necessary to use advanced genomic approaches to elucidate the molecular mechanisms underlying the aggressiveness of HNSCC cells. Analysis of our microRNA (miRNA) expression signature by RNA sequencing showed that the miR‐199 family (miR‐199a‐5p, miR‐199a‐3p, miR‐199b‐5p and miR‐199b‐3p) was significantly reduced in cancer tissues. Ectopic expression of mature miRNA demonstrated that all members of the miR‐199 family inhibited cancer cell migration and invasion by HNSCC cell lines (SAS and HSC3). These findings suggested that both passenger strands and guide strands of miRNA are involved in cancer pathogenesis. In silico database and genome‐wide gene expression analyses revealed that the gene coding for integrin α3 (ITGA3) was regulated by all members of the miR‐199 family in HNSCC cells. Knockdown of ITGA3 significantly inhibited cancer cell migration and invasion by HNSCC cells. Moreover, overexpression of ITGA3 was confirmed in HNSCC specimens, and high expression of ITGA3 predicted poorer survival of the patients (P = 0.0048). Our data revealed that both strands of pre‐miR‐199a (miR‐199a‐5p and miR‐199a‐3p) and pre‐miR‐199b (miR‐199b‐5p and miR‐199b‐3p) acted as anti‐tumor miRNA in HNSCC cells. Importantly, the involvement of passenger strand miRNA in the regulation of cellular processes is a novel concept in RNA research. Novel miRNA‐based approaches for HNSCC can be used to identify potential targets for the development of new therapeutic strategies.     

p21(Waf1/Cip1) and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells

We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.     

The histone deacetylase inhibitor PXD101 increases the efficacy of irinotecan in in vitro and in vivo colon cancer models

Purpose

Histone deacetylase inhibitors (HDACIs), such as PXD101 and suberoylanilide hydroxamic acid, inhibit proliferation and stimulate apoptosis of tumor cells. The enhanced effectiveness of chemotherapy or radiotherapy when combined with HDACIs has been observed in several cancers. In this study, we investigated the antitumor effect of PXD101 combined with irinotecan in colon cancer.

Methods

HCT116 and HT29 colon cancer cells for cell viability assay were treated with PXD101 and/or SN-38, the active form of irinotecan. Antitumor effects of HCT116 and HT29 xenografts treated with these combinations were evaluated. [¹⁸F]FLT-PET was used to detect early responses to PXD101 and irinotecan in colon cancer.

Results

PXD101 and SN38 possessed dose-dependent antiproliferative activity against HCT116 and HT29 cells and exerted a synergistic effect when used in combination. In xenografted mice, PXD101 in combination with irinotecan dramatically inhibited tumor growth without causing additive toxicity. Apoptotic effects on xenograft tumors were greater with combined treatment than with irinotecan alone. [¹⁸F]FLT-PET imaging revealed a 64% decrease in [¹⁸F]FLT uptake in tumors of HCT116 xenograft-bearing mice treated with a combination of PXD101 and irinotecan, indicating a decrease in thymidine kinase 1 (TK1) activity. These results were supported by Western blot analyses showing a decrease in tumor thymidine kinase 1 protein levels, suggesting that [¹⁸F]FLT-PET can be used to non-invasively detect early responses to these agents.

Conclusions

These data show that PXD101 increases the cytotoxic activity of irinotecan in in vitro and in vivo colon cancer models and suggest these agent combinations should be explored in the treatment of colon cancer.     

Neurotensin and a non‐peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10^–10 to 10^–6 M concentration range (ED₅₀ = 0.47 nM) while the neurotensin fragment (1–11) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54μmol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 μmol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22‐day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood‐borne neurotensin and suggest that non‐peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer. Int. J. Cancer 80:448–454, 1999. © 1999 Wiley‐Liss, Inc.     

MiR‐1285‐5p/TMEM194A axis affects cell proliferation in breast cancer

The onset of breast cancer among young patients is a major issue in cancer etiology. Our previous study has shown that poor prognosis in young women with breast cancer is associated with lower expression of the microRNA miR‐1285‐5p. In this study, we showed that the expression of miR‐1285‐5p is lower in tumor tissues than in normal tissues. Accumulating evidence suggests that miR‐1285‐5p plays critical roles in various types of cancers. However, the functional role of miR‐1285‐5p in breast cancer remains to be elucidated. Here, we showed the tumor‐suppressive role of miR‐1285‐5p and detailed its mechanism of action in breast cancer. Overexpression of miR‐1285‐5p significantly inhibited cell proliferation in breast cancer cells regardless of the tumor subtype. Among the target genes of miR‐1285‐5p, we found that transmembrane protein 194A (TMEM194A) was directly regulated by miR‐1285‐5p. Notably, separation of centrosomes from the nuclear envelope was observed upon knockdown of TMEM194A or overexpression of miR‐1285‐5p. In conclusion, our findings show that miR‐1285‐5p is a tumor suppressor via TMEM194A inhibition in breast cancer.     

MKK7 mediates miR‐493‐dependent suppression of liver metastasis of colon cancer cells

The prognosis of advanced colon cancer patients is profoundly affected by the presence or absence of liver metastasis. miR‐493 functions as a potent suppressor of liver metastasis, and low‐level miR‐493 expression in human primary colon cancer is associated with an elevated incidence of liver metastasis. We previously showed that IGF1R is a target gene of miR‐493, and that the inhibition of IGF1R partly explains how miR‐493 suppresses liver metastasis. However, major functional targets that mediate the antimetastatic activity of miR‐493 remain elusive. Here, we extended our search for target genes and identified MKK7, a mitogen‐activated protein kinase kinase, as a novel target of miR‐493. miR‐493 inhibits MKK7 expression by targeting the binding site at the 3′‐UTR of the mkk7 gene. MKK7 was expressed in six out of seven colon cancer cell lines examined but not in non‐transformed colon epithelial cells, and its expression was required for the activating phosphorylation of JNK. RNA interference‐mediated inhibition of MKK7 resulted in marked suppression of liver metastasis of colon cancer cells. A significant decrease of metastasized cells by the MKK7 knockdown was observed, even at early stages of the metastatic settlement, in accordance with a time course of the miR‐493‐mediated inhibition of the metastasis. Immunohistochemical examination in human primary colon tumors revealed that the occurrence of liver metastasis is associated with elevated levels of MKK7. Thus, MKK7 is a major functional target of miR‐493, and its suppression thwarts liver metastasis of colon cancer cells.     

Tumor‐suppressive microRNA‐223 inhibits cancer cell migration and invasion by targeting ITGA3/ITGB1 signaling in prostate cancer

Analysis of microRNA (miRNA) expression signatures in prostate cancer (PCa) and castration‐resistant PCa has revealed that miRNA‐223 is significantly downregulated in cancer tissues, suggesting that miR‐223 acts as a tumor‐suppressive miRNA by targeting oncogenes. The aim of this study was to investigate the functional roles of miR‐223 and identify downstream oncogenic targets regulated by miR‐223 in PCa cells. Functional studies of miR‐223 were carried out to investigate cell proliferation, migration, and invasion using PC3 and PC3M PCa cell lines. Restoration of miR‐223 significantly inhibited cancer cell migration and invasion in PCa cells. In silico database and genome‐wide gene expression analyses revealed that ITGA3 and ITGB1 were direct targets of miR‐223 regulation. Knockdown of ITGA3 and ITGB1 significantly inhibited cancer cell migration and invasion in PCa cells by regulating downstream signaling. Moreover, overexpression of ITGA3 and ITGB1 was observed in PCa clinical specimens. Thus, our data indicated that downregulation of miR‐223 enhanced ITGA3/ITGB1 signaling and contributed to cancer cell migration and invasion in PCa cells. Elucidation of the molecular pathways modulated by tumor‐suppressive miRNAs provides insights into the mechanisms of PCa progression and metastasis.     

Combinatorial inhibition of Plk1 and PKCβ in cancer cells with different p53 status

PKCβ and Plk1 are fascinating targets in cancer therapy. Therefore, we combined Enzastaurin targeting PKCβ and SBE13 targeting Plk1 to test synergistic effects in cells with different p53 status. We analyzed cell proliferation and apoptosis induction, and did Western blot and FACScan analyses to examine the combined PKCβ and Plk1 inhibition. p53-wild-type cells are more resistant to the combinatorial treatment than p53-deficient cells, which displayed a synergistic reduction of cell proliferation after the combination. HeLa, MCF-7 and HCT116^p53wt and HCT116^p53-/- cells differed in their cell cycle distribution after combinatorial treatment in dependence on a functional p53-dependent G1/S checkpoint (p53-deficient cells showed an enrichment in S and G2/M, p53-wild-type cells in G0/G1 phase). hTERT-RPE1 cells did not show the synergistic effects of cancer cells.Thus, we demonstrate for the first time that Plk1 inhibition using SBE13 enhances the effects of Enzastaurin in cancer cells. HCT116^p53wt and HCT116^p53-/- cells confirmed the p53-dependence of different effects after Plk1 and PKCβ inhibition observed in HeLa and MCF-7 cells. Obviously, p53 protects cells from the cytotoxicity of Enzastaurin in combination with SBE13. For that reason this combination can be useful to treat p53-deficient cancers, without displaying toxicity to normal cells, which all have functional p53.     

Sprouty2 protein enhances the response to gefitinib through epidermal growth factor receptor in colon cancer cells

Sprouty2 (Spry2) is known to increase the expression of epidermal growth factor receptors (EGFR) by conjugating with c‐Casitas B‐lineage lymphoma (C‐Cbl) to decrease protein degradation. The effect of Spry2 on the treatment of gefitinib, a tyrosine kinase inhibitor of EGFR, with regards to colon cancer is still unclear. The half maximal inhibitory concentration (IC50) values of gefitinib in six colon cancer cell lines were assessed. HCT116 and C2BBel cells expressed lower levels of Spry2 protein and were less sensitive to gefitinib, whereas HT29 cells that expressed high levels of Spry2 protein were more sensitive to gefitinib. The sensitivity to gefitinib was increased after overexpression of Spry2 in HCT116 cells, whereas it was decreased after Spry2 knockdown in HT29 cells. The levels of both phosphorylated and total EGFR were increased when HCT116 cells ectopically overexpressed Spry2, with concomitant increase in phosphatase and tensin homolog (PTEN) expression. Inhibition of EGFR by cetuximab reduced sensitivity to gefitinib in HCT116 cells overexpressing Spry2. However, knockdown of PTEN or K‐ras failed to diminish the effect of Spry2 on gefitinib sensitivity. Of note, Spry2 enhanced the antitumor effect of gefitinib in a xenograft model of HCT116 tumors, which harbored K‐ras codon 13 mutation. In conclusion, Spry2 can enhance the response of colon cancer cells to gefitinib by increasing the expression of phosphorylated and total EGFR. These results suggest that Spry2 may be a potential biomarker in predicting the response to anti‐EGFR treatment in colon cancer and that it is necessary to conduct clinical studies to incorporate Spry2 into the network of cancer treatment. (Cancer Sci 2010)     

Crucial involvement of the CCL3‐CCR5 axis‐mediated fibroblast accumulation in colitis‐associated carcinogenesis in mice

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis‐associated cancer in mice. AOM/DSS‐induced tumor formation was reduced in CCL3‐ or its specific receptor, CCR5‐deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS‐induced type I collagen‐positive fibroblast accumulation in the colon was reduced in CCL3‐ or CCR5‐deficient mice. This was associated with depressed expression of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB‐EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB‐EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3‐CCR5‐mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full‐blown colitis‐associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis‐associated cancer.