Signaling and regulation of transcription factor nuclear factor-kappaB (NF-B) has been an area of extensive research since its first discovery nearly three decades ago. Members of the NF-B family have been reported to critically mediate a multitude of responses in normal cells. Therefore, it is not surprising that NF-B function can go awry and result in pathological conditions including cancer. Despite its critical importance, the functional role of NF-B has not received the same attention in cancers of all tissue types. In the case of cancer of the urinary bladder, which is the second most common urologic cancer, the involvement of NF-B in the development of superficial or muscle invasive disease and during cancer recurrence is rudimentary at best. Nuclear expression of p65/RelA is seen in bladder cancer patients and has been found to negatively affect survival of patients with superficial and muscle invasive disease. Despite these observations, the exact mechanism of NF-B upregulation and function remains unknown. Furthermore, the emergence of a tumor suppressive role for NF-B in recent years suggests that the family may play the role of a double-edged sword in cancer, which remains unexplored in bladder cancer. The challenge now is to delineate the increasing complexity of this pathway in the development and progression of bladder cancer. Here, we review key aspects of the current knowledge of signaling and regulation by the NF-B family focusing on its controversial role in cancer and highlight the importance of studying NF-B in bladder cancer in particular.
Radiogenomics is the whole genome application of radiogenetics, which focuses on uncovering the underlying genetic causes of individual variation in sensitivity to radiation. There is a growing consensus that radiosensitivity is a complex, inherited polygenic trait, dependent on the interaction of many genes involved in multiple cell processes. An understanding of the genes involved in processes such as DNA damage response and oxidative stress response, has evolved toward examination of how genetic variants, most often, single nucleotide polymorphisms (SNPs), may influence interindividual radioresponse. Many experimental approaches, such as candidate SNP association studies, genome-wide association studies and massively parallel sequencing are being proposed to address these questions. We present a review focusing on recent advances in association studies of SNPs to radiotherapy response and discuss challenges and opportunities for further studies. We also highlight the clinical perspective of radiogenomics in the future of personalized treatment in radiation oncology.
Aberrant activation of Wnt signaling pathway is crucial for the onset and progression of human colorectal cancer (CRC). Owing to the persistent dependence on Wnt signaling for growth and survival, inhibition of this pathway is an attractive approach for new therapies. 11α, 12α-epoxyleukamenin E (EPLE) is a novel ent-kaurane diterpenoid that we previously isolated from Salvia cavaleriei, exhibiting antitumor activities in a variety of cancer cells. Herein, we found that whereas sparing normal human colon mucosal epithelial cells, EPLE selectively inhibited the proliferation of CRC cell lines as well as primary tumor cells. Mechanistically, we demonstrated EPLE exerted its function through suppressing Wnt signaling pathway, as evidenced by EPLE-mediated downregulation of Wnt target genes such as c-Myc, Axin2 and Survivin. Consistently, luciferase reporter assays showed that the EPLE directly blocked Wnt/β-catenin–mediated transcriptional activation. In combination of co-immunoprecipitation and protein structure-based analyses, we determined that the EPLE entered the interface of β-catenin/TCF4 complexes and blocked their interaction that is required for β-catenin–mediated transcriptional activation. Moreover, overexpression of β-catenin alleviated the cytotoxicity of EPLE in CRC cells, supporting Wnt signaling is a major and specific target of EPLE. Combined treatments of EPLE and 5-fluorouracil, the first-line chemotherapy for CRC patients, achieved a synergistic effect. More importantly, EPLE hampered tumor development in a CRC xenograft model. Our data thus establish EPLE as a novel inhibitor of Wnt signaling that holds great promise as a potential candidate for further preclinical evaluation for CRC treatments.
Epithelial–mesenchymal transition (EMT) is an important process implicated in tumor invasion and metastasis. Twist1 is a transcription factor that induces EMT, including E-cadherin suppression and cancer cell migration and invasion; hence it promotes cancer metastasis. Twist1 directly or indirectly regulates the expression of various genes and cellular functions involved in cancer progression. However, the underlying mechanisms remain largely unknown. In this study, we investigated the molecular basis for Twist1-mediated invasion and EMT. In human cancer cells, Twist1 was found to directly upregulate transcription of the mesenchymal gene integrin α5 in an E-box-independent, but activating protein-1 (AP-1) element-dependent, manner. Twist1 activated the integrin α5 promoter by interacting with and activating the transcription factor AP-1, composed of c-Jun and activating transcription factor-2 (ATF-2); it also enhanced the nuclear presence of ATF-2. AP-1 was critical for Twist1-induced cancer cell invasion, primarily through the induction of integrin α5, which activated c-Jun N-terminal kinase and focal adhesion kinase-signaling activities. Using data from The Cancer Genome Atlas, we found that Twist1 expression positively correlates with integrin α5 expression in human colorectal cancers. These findings suggest that cooperation between Twist1 and AP-1 represents a novel mechanism for EMT and tumor invasiveness. This study supports further investigation into the molecular basis underlying the diverse Twist1-mediated functions that occur during tumor progression.
MicroRNA-7 (miR-7) has been reported to be a tumor suppressor in all malignancies including colorectal cancer (CRC). However, its significance for CRC clinical outcomes has not yet been explored. The potential for miR-7 to act as a tumor suppressor by coordinately regulating the epidermal growth factor receptor (EGFR) signaling pathway at several levels was examined. We investigated the tumor inhibitory effect of miR-7 in CRC, with particular focus on the relationship between miR-7 and the EGFR pathway. Quantitative reverse transcription–PCR was used to evaluate miR-7 expression in 105 CRC cases to determine the clinicopathologic significance of this miRNA. The regulation of EGFR by miR-7 was examined with miR-7 precursor-transfected cells. Furthermore, we investigated whether miR-7 suppresses proliferation of CRC cells in combination with cetuximab, a monoclonal antibody against EGFR. Multivariate analysis indicated that low miR-7 expression was an independent prognostic factor for poor survival (P = 0.0430). In vitro assays showed that EGFR and RAF-1 are direct targets of miR-7, which potently suppressed the proliferation of CRC cells, and, interestingly, that the growth inhibitory effect of each of these was enhanced by cetuximab. miR-7 is a meaningful prognostic marker. Furthermore, these data indicate that miR-7 precursor, alone or in combination with cetuximab, may be useful in therapy against CRC.
Rac GTPase-activating protein (RacGAP) 1 plays a key role in controlling various cellular phenomena including cytokinesis, transformation, invasive migration and metastasis. This study investigated the function and clinical significance of RacGAP1 expression in colorectal cancer (CRC). The intrinsic functions of RacGAP1 in CRC cells were analyzed using small interfering RNA (siRNA). We analyzed RacGAP1 mRNA expression in surgical specimens from 193 CRC patients (Cohort 1) by real-time PCR. Finally, we validated RacGAP1 protein expression using formalin-fixed paraffin-embedded samples from 298 CRC patients (Cohort 2) by immunohistochemistry. Reduced RacGAP1 expression by siRNA in CRC cell lines showed significantly decreased cellular proliferation, migration and invasion. In Cohort 1, RacGAP1 expression in CRC was significantly higher than in adjacent normal mucosa and increased according to tumor node metastasis stage progression. High RacGAP1 expression in tumors was significantly associated with progression and prognosis. In Cohort 2, RacGAP1 protein was overexpressed mainly in the nuclei of CRC cells; however, its expression was scarcely observed in normal colorectal mucosa. RacGAP1 protein expression was significantly higher in CRC patients with higher T stage, vessel invasion and lymph node and distant metastasis. Increased expression of RacGAP1 protein was significantly associated with poor disease-free and overall survival. Multivariate analyses revealed that high RacGAP1 expression was an independent predictive marker for lymph node metastasis, recurrence and poor prognosis in CRC. Our data provide novel evidence for the biological and clinical significance of RacGAP1 as a potential biomarker for identifying patients with lymph node metastasis and poor prognosis in CRC.
Resistance to cytotoxic chemotherapy is a major cause of mortality in colorectal cancer (CRC) patients. Chemoresistance has been linked primarily to a subset of cancer cells undergoing epithelial–mesenchymal transition (EMT). Curcumin, a botanical with antitumorigenic properties, has been shown to enhance sensitivity of cancer cells to chemotherapeutic drugs, but the molecular mechanisms underlying this phenomenon remain unclear. Effects of curcumin and 5-fluorouracil (5FU) individually, and in combination, were examined in parental and 5FU resistant (5FUR) cell lines. We performed a series of growth proliferation and apoptosis assays in 2D and 3D cell cultures. Furthermore, we identified and analyzed the expression pattern of a subset of putative EMT-suppressive microRNAs (miRNAs) and their downstream target genes regulated by curcumin. Chemosensitizing effects of curcumin were validated in a xenograft mouse model. Combined treatment with curcumin and 5FU enhanced cellular apoptosis and inhibited proliferation in both parental and 5FUR cells, whereas 5FU alone was ineffective in 5FUR cells. A group of EMT-suppressive miRNAs were upregulated by curcumin treatment in 5FUR cells. Curcumin suppressed EMT in 5FUR cells by downregulating BMI1, SUZ12 and EZH2 transcripts, key mediators of cancer stemness-related polycomb repressive complex subunits. Using a xenograft and mathematical models, we further demonstrated that curcumin sensitized 5FU to suppress tumor growth. We provide novel mechanistic evidence for curcumin-mediated sensitization to 5FU-related chemoresistance through suppression of EMT in 5FUR cells via upregulation of EMT-suppressive miRNAs. This study highlights the potential therapeutic usefulness of curcumin as an adjunct in patients with chemoresistant advanced CRC.
Lung cancer is a leading cause of death in developed countries. Although smoking cessation is a primary strategy for preventing lung cancer, former smokers remain at high risk of cancer. Accordingly, there is a need to increase the efficacy of lung cancer prevention. Poor bioavailability is the main factor limiting the efficacy of chemopreventive agents. The aim of this study was to increase the efficacy of cancer chemopreventive agents by using lipid nanoparticles (NPs) as a carrier. This study evaluated the ability of lipid NPs to modify the pharmacodynamics of chemopreventive agents including N-acetyl-L-cysteine, phenethyl isothiocyanate and resveratrol (RES). The chemopreventive efficacy of these drugs was determined by evaluating their abilities to counteract cytotoxic damage (DNA fragmentation) induced by cigarette smoke condensate (CSC) and to activate protective apoptosis (annexin-V cytofluorimetric staining) in bronchial epithelial cells both in vitro and in ex vivo experiment in mice. NPs decreased the toxicity of RES and increased its ability to counteract CSC cytotoxicity. NPs significantly increased the ability of phenethyl isothiocyanate to attenuate CSC-induced DNA fragmentation at the highest tested dose. In contrast, this potentiating effect was observed at all tested doses of RES, NPs dramatically increasing RES-induced apoptosis in CSC-treated cells. These results provide evidence that NPs are highly effective at increasing the efficacy of lipophilic drugs (RES) but are not effective towards hydrophilic agents (N-acetyl-L-cysteine), which already possess remarkable bioavailability. Intermediate effects were observed for phenethyl isothiocyanate. These findings are relevant to the identification of cancer chemopreventive agents that would benefit from lipid NP delivery.
To elucidate the function of the Ras-homologous GTPase Rac1 in hepatocarcinogenesis induced by diethylnitrosamine (DEN), mice lacking hepatic Rac1 expression were treated with DEN and compared to the wild-type (WT). Rac1 knock-out (KO) mice were found to have a lower tumor yield as compared to Rac1 proficient mice. The small-sized tumors formed in the absence of Rac1 lack an activated Ras/Raf/mitogen-activated protein kinase pathway, as indicated by the absence of p-ERK expression. Apparently, Rac1 is required for Ras-driven oncogenic pathways. Moreover, tumors in Rac1 deficient mice were glutamine synthase (GS) negative. They displayed a high number of p-H3-positive and cyclinB1 expressing cells, pointing to a defect in mitotic progression. To elucidate the influence of Rac1 on mechanisms of tumor initiation, acute DEN-induced hepatic stress responses were monitored. Rac1 deficiency caused fairly complex, partially time-dependent, alterations in both basal and/or DEN-induced messenger RNA (mRNA) and protein levels of susceptibility-related genes. Basal protein expression of DNA repair factors Brca1 and DNA repair protein RAD51 homolog (Rad51) and the cell cycle regulatory factor p27 was enhanced in the absence of Rac1. Following DEN treatment, p21 mRNA and protein expression was stimulated independent of the Rac1 status. Lack of Rac1 increased mechanisms of the DNA damage response (DDR), as shown by elevated protein levels of p-ATR, p-p53 and H2AX 24h after DEN treatment. The data show that Rac1 is essential for DEN-stimulated hepatocarcinogenesis. We hypothesize that it promotes tumor initiation by counteracting the elimination of initiated cells and, moreover, alleviates the outgrowth of transformed cells. Hence, pharmacological targeting of Rac1 could be suitable for chemoprevention.
MicroRNAs (miRNAs) are thought to have an important role in tumor metastasis by regulating diverse cellular pathways. Here, we describe the function and regulation network of miR-206 in gastric cancer (GC) metastasis. MiR-206 expression was downregulated in GC cells especially in high metastatic potential cells and was also significantly decreased in metastatic lesions compared with their corresponding primary tumor samples. Both gain- and loss-of-function studies confirmed that miR-206 significantly suppressed GC cell invasion and metastasis both in vitro and in vivo. Mechanistically, paired box gene 3 (PAX3) was identified as a functional target of miR-206 in GC cells. MiR-206 inhibited GC metastasis by negatively regulating expression of PAX3. In addition, PAX3 expression was markedly higher in GC tissues than in adjacent non-cancerous tissues. GC patients with positive PAX3 expression had shorter overall survival times. Transwell assays and in vivo metastasis assays demonstrated that overexpression of PAX3 significantly promoted the invasiveness and pulmonary metastasis of GC cells. On the other hand, downregulation of PAX3 markedly reduced cell metastatic potential. Mechanistic investigations indicated that prometastasis function of PAX3 was mediated by upregulating downstream target MET. Moreover, we found that levels of PAX3 and MET were positively correlated in matched human GC specimens, and their coexpression was associated with poor prognoses. In conclusion, our results reveal that miR-206-PAX3-MET signaling is critical to GC metastasis. Targeting the pathway described here may open new therapeutic prospects to restrict the metastatic potential of GC.
Salicylic acid and anthocyanidins are known as plant-derived antioxidants, but also can provoke paradoxically seeming prooxidant effects in vitro. These prooxidant effects are connected to the potential of salicylic acid and anthocyanidins to induce apoptosis selectively in tumor cells in vitro and to inhibit tumor growth in animal models. Several epidemiological studies have shown that salicylic acid and its prodrug acetylsalicylic acid are tumor-preventive for humans. The mechanism of salicylic acid- and anthocyanidin-dependent antitumor effects has remained enigmatic so far. Extracellular apoptosis-inducing reactive oxygen species signaling through the NO/peroxynitrite and the HOCl signaling pathway specifically induces apoptosis in transformed cells. Tumor cells have acquired resistance against intercellular reactive oxygen species signaling through expression of membrane-associated catalase. Here, we show that salicylic acid and anthocyanidins inactivate tumor cell protective catalase and thus reactive apoptosis-inducing intercellular reactive oxygen species signaling of tumor cells and the mitochondrial pathway of apoptosis Salicylic acid inhibits catalase directly through its potential to transform compound I of catalase into the inactive compound II. In contrast, anthocyanidins provoke a complex mechanism for catalase inactivation that is initiated by anthocyanidin-mediated inhibition of NO dioxygenase. This allows the formation of extracellular singlet oxygen through the reaction between H2O2 and peroxynitrite, amplification through a caspase8-dependent step and subsequent singlet oxygen-mediated inactivation of catalase. The combination of salicylic acid and anthocyanidins allows for a remarkable synergistic effect in apoptosis induction. This effect may be potentially useful to elaborate novel therapeutic approaches and crucial for the interpretation of epidemiological results related to the antitumor effects of secondary plant compounds.