Chongwen Xu

Smac: Its role in apoptosis induction and use in lung cancer diagnosis and treatment

Apoptosis is a conserved and regulated cell suicide process, the malfunction of which is closely linked with carcinogenesis. Caspases control the induction of apoptosis through an enzymatic cascade that can be activated by both the mitochondrial and death receptor pathways. Smac is a mitochondrial protein that interacts with Inhibitor of Apoptosis Proteins (IAPs) and, upon apoptotic stimuli, is released into the cytoplasm to inhibit the capase-binding activity of IAPs. Smac plays key roles in both the diagnosis and treatment of cancer, especially lung cancer. Our review will focus on the roles of Smac in lung carcinogenesis and cancer progression and its relevance in lung cancer treatment.

The insights of Let-7 miRNAs in oncogenesis and stem cell potency

The ability of the classic tumour‐suppressive let‐7 family to inhibit carcinogenesis, tumour progression, recurrence and pluripotency of cancer stem cells has generated significant interest in the field of cancer research. Through suppressing and degrading downstream‐targeted mRNAs, let‐7 affected most aspects of cell biology. It is perplexing how let‐7 affects oncogenesis, as the large influx of new miRNAs and other kinds of non‐coding RNAs are continuously defined. In this review, we delineate the complex functions of let‐7 and discuss the future direction of let‐7 research.

DICER1 regulated let‐7 expression levels in p53‐induced cancer repression requires cyclin D1

Let‐7 miRNAs act as tumour suppressors by directly binding to the 3′UTRs of downstream gene products. The regulatory role of let‐7 in downstream gene expression has gained much interest in the cancer research community, as it controls multiple biological functions and determines cell fates. For example, one target of the let‐7 family is cyclin D1, which promotes G0/S cell cycle progression and oncogenesis, was correlated with endoribonuclease DICER1, another target of let‐7. Down‐regulated let‐7 has been identified in many types of tumours, suggesting a feedback loop may exist between let‐7 and cyclin D1. A potential player in the proposed feedback relationship is Dicer, a central regulator of miRNA expression through sequence‐specific silencing. We first identified that DICER1 is the key downstream gene for cyclin D1‐induced let‐7 expression. In addition, we found that let‐7 miRNAs expression decreased because of the p53‐induced cell death response, with deregulated cyclin D1. Our results also showed that cyclin D1 is required for Nutlin‐3 and TAX‐induced let‐7 expression in cancer repression and the cell death response. For the first time, we provide evidence that let‐7 and cyclin D1 form a feedback loop in regulating therapy response of cancer cells and cancer stem cells, and importantly, that alteration of let‐7 expression, mainly caused by cyclin D1, is a sensitive indicator for better chemotherapies response.

MiR-208a stimulates the cocktail of SOX2 and β-catenin to inhibit the let-7 induction of self-renewal repression of breast cancer stem cells and formed miR208a/let-7 feedback loop via LIN28 and DICER1

MiR-208a stimulates cardiomyocyte hypertrophy, fibrosis and β-MHC (β-myosin heavy chain) expression, being involved in cardiovascular diseases. Although miR-208a is known to play a role in cardiovascular diseases, its role in cancer and cancer stem cells (CSCs) remains uncertain. We identified an inverse relationship between miR-208a and let-7a in breast cancer specimens, and found that SOX2, β-catenin and LIN28 are highly expressed in patients with advanced breast cancer opposed to lesser grades. Further, we isolated ALDH1+ CSCs from ZR75–1 and MDA-MB-231 (MM-231) breast cancer cell lines to test the role of miR-208a in breast CSCs (BrCSCs). Our studies showed that overexpression of miR-208a in these cells strongly promoted the proportion of ALDH1+ BrCSCs and continuously stimulated the self-renewal ability of BrCSCs. By using siRNAs of SOX2 and/or β-catenin, we found that miR-208a increased LIN28 through stimulation of both SOX2 and β-catenin. The knockdown of either SOX2 or β-catenin only partially attenuated the functions of miR-208a. Let-7a expression was strongly inhibited in miR-208a overexpressed cancer cells, which was achieved by miR-208a induction of LIN28, and the restoration of let-7a significantly inhibited the miR-208a induction of the number of ALDH1+ cells, inhibiting the propagations of BrCSCs. In let-7a overexpressed ZR75–1 and MM-231 cells, DICER1 activity was significantly inhibited with decreased miR-208a. Let-7a failed to decrease miR-208a expression in ZR75–1 and MM-231 cells with DICER1 knockdown. Our research revealed the mechanisms through which miR-208a functioned in breast cancer and BrCSCs, and identified the miR-208a-SOX2/β-catenin-LIN28-let-7a-DICER1 regulatory feedback loop in regulations of stem cells renewal.

Overexpression of Smac Promotes Cisplatin-Induced Apoptosis by Activating Caspase-3 and Caspase-9 in Lung Cancer A549 Cells

Second mitochondrial-derived activator of caspase (Smac) plays crucial roles in mitochondrial apoptosis pathways and promotes chemotherapy-induced apoptosis. In this study, Smac levels were examined in various lung cancer cell lines, and the effects of overexpressed Smac in the nonsmall-cell lung cancer cell line A549 were assayed by stable transfection of Smac. Subsequently, MTT assays, cell counting, and flow cytometry were applied to show that overexpression of Smac inhibits cell viability and cell growth and enhances apoptosis after cisplatin treatment. Western blotting was performed before and after cisplatin treatment to demonstrate that drug treatment could release Smac from mitochondria into the cytosol and promote apoptosis by activating caspase-3 and caspase-9. Promotion of apoptosis by cytosolic Smac could be blocked by pretreating cells with the caspase-9 inhibitor z-LEHD-FMK. Our findings indicate that overexpressed Smac significantly inhibited A549 cell growth and promoted apoptosis following cisplatin treatment due to the release of Smac from mitochondria into the cytosol, which increased the activities of caspase-3 and caspase-9.

Let-7a regulates mammosphere formation capacity through Ras/NF-κB and Ras/MAPK/ERK pathway in breast cancer stem cells

Breast cancer stem cells (BCSCs) have the greatest potential to maintain tumorigenesis in all subtypes of tumor cells and were regarded as the key drivers of tumor. Recent evidence has demonstrated that BCSCs contributed to a high degree of resistance to therapy. However, how BCSCs self renewal and tumorigenicity are maintained remains obscure. Herein, our study illustrated that overexpression of let-7a reduced cell proliferation and mammosphere formation ability of breast cancer stem cells(BCSCs) in a KRas-dependent manner through different pathways in vitro and in vivo. To be specific, we provided the evidence that let-7a was decreased, and reversely the expression of KRas was increased with moderate expression in early stages (I/II) and high expression in advanced stages (III/IV) in breast cancer specimens. In addition, the negative correlation between let-7a and KRas was clearly observed. In vitro, we found that let-7a inhibited mammosphere-forming efficiency and the mammosphere-size via NF-κB and MAPK/ERK pathway, respectively. The inhibitory effect of let-7a on mammosphere formation efficiency and the size of mammospheres was abolished after the depletion of KRas. On the contrary, enforced expression of KRas rescued the effect of let-7a. In vivo, let-7a inhibited the growth of tumors, whereas the negative effect of let-7a was rescued after overexpressing KRas. Taken together, our findings suggested that let-7a played a tumor suppressive role in a KRas-dependent manner.

Hyperthermia induces apoptosis by targeting Survivin in esophageal cancer

Hyperthermia is considered the fifth pillar of cancer treatment. It induces cancer cell apoptosis, however, its molecular mechanisms remain unclear. In the present study, the role of Survivin in hyperthermia-induced apoptosis in esophageal cancer was investigated. Different temperatures were used to treat EC109 esophageal cancer cells, and their viability was found to be significantly inhibited with a concomitant increase in apoptosis and necrosis. Necrosis increased in a temperature‑dependent manner, whereas peak apoptosis was reached at 43˚C. The hyperthermia-induced apoptosis was due to the inhibition of Survivin and the activation of caspase-3. Subsequently, overexpression of Survivin inhibited the activation of caspase-3 and hyperthermia-induced apoptosis, however, this inhibition was reversed in the absence of XIAP. Immunoprecipitations showed that Survivin did not directly bind to caspase-3, whereas XIAP interacted with Survivin and caspase-3. Immunohistochemistry was performed to detect the expression of Survivin in esophageal cancer patient samples. A higher expression of Survivin in esophageal cancer tissues compared to normal tissues was observed, and a high expression correlated with poor prognosis. The results indicated that hyperthermia decreases the expression of Survivin, prevents its binding to XIAP, activates caspase-3 and induces apoptosis. Due to its correlation with poor prognosis, Survivin may be a target for hyperthermia in the treatment of esophageal cancer.

Nano Let‑7b sensitization of eliminating esophageal cancer stem‑like cells is dependent on blockade of Wnt activation of symmetric division

The poor therapy response and poor prognosis of esophageal cancer has made it one of the most malignant carcinoma, and the complicated multidisciplinary treatment failed to achieve a long-term disease-free survival. To diagnose esophageal cancer at an earlier stage, and to improve the effect of anticancer therapy would improve the therapeutic efficacy. After retrospective analysis of the cancer samples of patients who received esophagectomy, we found the relevance between ratio of either ALDH1 or CD133-positive cancer stem cells and 2-year recurrence. Higher ratios of cancer stem cells indicated later clinical stages, and Wnt signaling activation was more frequent in later esophageal carcinoma. Further in bench studies, we explored the suppressive roles and the mechanisms involved in Let-7 on self-renewal in ECA-109 and ECA-9706 esophageal cancer stem cells. Isolated cancer stem cells naturally divide symmetrically and are therapy resistant. Therapy of fluorouracil and docetaxel both enriched the stem cells, proving the resistant characteristics of cancer stem cells. Wnt activation stimulated more symmetric division of stem cells, resulting in self-renewal promotion, which could be blocked by Let-7 overexpression. Furthermore, enforced Let-7 sensitized the stem cells to chemotherapies in a Wnt pathway inhibition-dependent manner, contributing to Let-7 sensitization of chemotherapeutic response. Wnt activation weakened the suppressive Let-7b through the sponge functions of CCAT-1, forming the negative feedback loop of Let-7b/Wnt/CCAT1. These results identified the crucial participation of stem cells in esophageal cancer occurrence and progression as the potent indicator, and also indicate the potential powerful agent of Let-7 nano-particles in treatment of cancer.

MiR-129 blocks estrogen induction of NOTCH signaling activity in breast cancer stem-like cells

Stem-like cells in tumor group featured the major role in the chemotherapy resistance of breast cancer, and the reduction of stem-like cells helped to perish the tumor when receiving chemotherapy. Smaller stem cells number indicated better therapeutic effect in vitro and in clinics, but how did miR-129 and Notch signaling function in breast cancer stem-like cells (BrCSCs) were unclear yet. Through using sphere forming assay and FACS sorting, we found that miR-129 decreased the proportion of stem-like cells in breast cancer cells. Results further indicated that miR-129 degraded the Estrogen Receptor 1 (ESR1) mRNA through a post-translational manner and contributed to the decline of stem-like cells number, preventing tumor regeneration. Cyclin d1 and DICER 1 were proved to promote Let-7 maturation, and in present study, we proved that miR-129 exhibited inhibition on ESR1 and halted the cyclin d1/DICER 1 sustaining of Let-7, which consequently released the Let-7 degradation of NUMB. The restoration of suppressive NUMB by upregulating miR-129 resulted in NOTCH signaling inhibition. In conclusion, we demonstrated the negative regulation of miR-129 on NOTCH signaling activation in BrCSCs’ renewal, which was achieved via continuous suppression on cyclin d1/DICER1 sustaining of Let-7 level, and eventually rescued the targeted inhibition of NUMB. The miR-129/ESR1 signaling played pivotal role in controlling DICER1/Let-7/NOTCH cascade via cyclin d1, revealing the novel mechanism of dual Let-7 in non-coding genes network.

Indomethacin induces apoptosis in the EC109 esophageal cancer cell line by releasing second mitochondria-derived activator of caspase and activating caspase-3

The use of non‑steroidal anti‑inflammatory drugs (NSAIDs) has been associated with a reduced risk of various types of cancer, including esophageal cancer. However, the mechanisms underlying the antineoplastic effects of NSAIDs in esophageal cancer remain to be elucidated. In the present study, a significant inhibition in cell viability was observed in the EC109 cells following treatment with different concentrations of indomethacin, and these effects occurred in a dose‑ and time‑dependent manner. This inhibition was due to the release of second mitochondria‑derived activator of caspase (Smac) into the cytosol and the activation of caspase‑3. Subsequently, flow cytometry was performed to investigate indomethacin‑induced apoptosis following the overexpression or knockdown of Smac, and western blot analysis was performed to determine the expression of Smac and the activation of caspase‑3. Overexpression of Smac was promoted apoptosis, while downregulation of Smac significantly inhibited apoptosis. Western blot analysis demonstrated that indomethacin induced apoptosis through releasing Smac into the cytosol and activating caspase‑3. These results indicated that Smac is essential for the apoptosis induced by indomethacin in esophageal cancer cells.