Ning Du

Angiotensin II receptor type 1 blockers suppress the cell proliferation effects of angiotensin II in breast cancer cells by inhibiting AT1R signaling

Chronic stress and a high-fat diet are well-documented risk factors associated with the renin-angiotensin system in the development of breast cancer. The angiotensin II type 1 receptor (AT1R) is a novel component of the renin-angiotensin system. Several recent studies have focused on the function of AT1R in cell proliferation during cancer development. Thus, we hypothesized that angiotensin II (Ang Ⅱ) can promote proliferation of breast cancer via activated AT1R; the activation of AT1R may play an important role in promoting breast cancer growth, and AT1R blocker (ARB) may suppress the promotional effect on proliferation by antagonizing AT1R. The expression level of AT1R was found to be significantly upregulated in breast cancer cells by immunohistochemistry, but no correlation between AT1R expression and ER/PR/Her-2 expression was observed. The AT1R(+)-MCF-7 cell line exhibited high expression of AT1R protein, and we generated the AT1R(-)-MCF-7 cell line using RNA interference. ARBs, and in particular irbesartan, effectively inhibited the effects of Ang II on cell proliferation, cell cycle development and downstream AT1R signaling events, including the activation of the Ras-Raf-MAPK pathway and the transcription factors NF-κB and CREB. Irbesartan also significantly altered p53, PCNA and cyclin D1 expression, which was also influenced by activated AT1R in AT1R(+)-MCF-7 cells. These results suggest that ARBs may be useful as a novel preventive and therapeutic strategy for treating breast cancer.

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.

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.

Possible carcinogenesis of tumor suppressor let-7

Cancer stem cells (CSCs), a group showing high capacities of sphere-forming and self renewal, are blamed for tumor initiation, recurrence and therapy resistance. Therefore, therapeutics specifically targeting and perishing CSCs may be promising. Let-7 miRNAs, one of the earliest discovered miRNAs, were considered as novel and vital agents to eliminate cancer and CSCs. However, in recent researches, many regulatory loops among let-7 and its targeted genes were noticed; the regulation of let-7 caused by its hunting mRNAs helped to form the hypotheses that hunters and preys may swap their roles when in confrontations. Besides, the evil side of let-7 was discovered occasionally, therefore, we hypothesize that dual characteristics of let-7 do exist, which will have significant impacts on anticancer research. Targeted therapies against cancer and CSCs by using let-7 or other miRNAs as weapons should be thought twice before clinical application.

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.

Tumor necrosis factor α stimulates Her-2 cleavage by activated caspase-8.

Background/Aim: Her-2 over-expression has been correlated with a poor prognosis in patients with breast cancer. Now, we explored the effect of TNF-α treatment and/or NFĸB activation on Her-2 expression in MCF-7 breast adenocarcinoma cells. Methods: Stably transfected MCF-7 cell lines with pcDNA3.0, IĸBα MT, c-FLIP/control shRNA were established by FuGENE with the supplementation of G418 (500 µg /ml). Western blot and Real-time PCR were applied to assess the expression levels of protein and mRNA of target gene. In addition, caspase-8 activity was evaluated by the incubation with a caspase-8 fluorogenic substrate, Ac-IEPD-AMC using a spectrofluorometer. Results: It was uncovered that Her-2 was a new substrate for caspase-8 and that tumor necrosis factor α (TNF-α) stimulation resulted in a caspase-8-dependent Her-2 cleavage in MCF-7 breast adenocarcinoma cells defective for nuclear factor ĸB (NFĸB) activation. We demonstrated that the antiapoptotic transcription factor NFĸB counteracted this cleavage through the induction of caspase-8 inhibitor, c-FLIP. Conclusion: we propose a novel mechanism in which NFĸB functions as a new antiapoptotic factor by counteracting TNF-α-triggered Her-2 cleavage.

Analysis of risk factors for post‑operative complications and prognostic predictors of disease recurrence following definitive treatment of patients with esophageal cancer from two medical centers in Northwest China

Evaluating the clinicopathological features of patients receiving definitive treatment for esophageal cancer may facilitate the identification of patterns and factors associated with post-operative complications, and enable the development of a surveillance strategy for surviving patients at a higher risk of disease recurrence. In the present study, clinical data from 579 patients with esophageal cancer that underwent radical resection of esophagus were collected. These patients were admitted to two medical centers in Northwest China, and information regarding the presence or absence of basic chronic diseases and post-operative results were retrospectively analyzed. The level of selected stem cell markers, including aldehyde dehydrogenase 1, CD133, integrin subunit α 6, integrin subunit β 4 and T-cell factor-4, were determined in esophageal cancer tissue samples in order to determine whether these markers may be useful predictors of disease prognosis and recurrence. Post-operative complications in patients receiving radical resection of the esophagus included respiratory system complications, cardiovascular abnormalities and esophageal anastomotic fistulae. Diabetes, basic respiratory disease and lower pre-surgical serum albumin levels were observed to be individual risk factors associated with post-operative complications, including respiratory system complications of acute respiratory failure and pulmonary infection, cardiovascular abnormalities of atrial fibrillation and arrhythmia, as well as the development of esophageal anastomotic fistulae. Diagnosis of esophageal cancer at later stage was significantly correlated with anastomotic fistula. Molecular detection of stem cell markers for prognosis prediction was achieved by immunohistochemical and immunofluorescence staining assays. The results demonstrated that the presence of stem-like cells in cancer tissues was associated with poor disease prognosis and a high recurrence ratio. In conclusion, the results of the current study suggested that post-operative complications were more likely to occur in patients with diabetes, basic respiratory disease or lower serum albumin levels prior to surgery. Therefore, sufficient intensive peri-operative care, rigorous operative risk assessments, and the selection of the patients with early or mid-stage esophageal cancer, may decrease the risk of post-surgical complications in patients receiving radical resection of the esophagus. In addition, a high ratio of esophageal cancer stem-like cells was associated with cancer recurrence. These results suggest that an intensive surveillance strategy should be implemented in order to facilitate early detection of disease recurrence and improve the clinical management of these patients post-surgery.

Clinical Application of Detecting 21-Gene Recurrence Score in Predicating Prognosis and Therapy Response of Patients with Breast Cancer from Two Medical Centers

ABSTRACT To determine the most suitable strategy in treating patients with invasive breast cancer from Northwest China. Lower recurrence score (RS) correlated with lower recurrence ratio. Patients having a medium-high 21-gene RS who received adjuvant therapy presented lower recurrence risk. Younger patients having RS results (⩾31) tended to accept adjuvant therapy more often, however, those having intermediate RS results were inclined to wait and did not receive chemotherapy. These results suggested that RS-based precision medicine will allow individualized diagnosis and treatment, resulting in better outcomes and preserved medical resources.