Xiaoting Jia

Low concentration of metformin induces a p53-dependent senescence in hepatoma cells via activation of the AMPK pathway

The induction of senescence for cancer treatment has provoked considerable interest recently. Metformin, a first-line drug for diabetes mellitus type 2, appears to be associated with a lower risk and improved outcomes in hepatocellular carcinoma (HCC). The mechanism involved in function of metformin in HCC is poorly understood. We show that low doses of metformin induced hepatoma cell senescence characterized by accumulation of senescence-associated β-galactosidase activity (SA-β-gal) and the senescence marker Dec1, whereas the higher doses initiated apoptotic cell death. Metformin-induced senescence was accompanied by enhanced phosphorylation levels of AMP-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase (ACC). The expression of acetylated p53 at Lys382 (Ac-p53) and p21 was also increased, while phosphorylation of p53 at Ser15 (p-p53), p53, p16 and pRB was rarely altered after metformin treatment. Moreover, inhibition of AMPK decreased p-AMPK, p-ACC, Ac-p53 and p21 expression, diminished SA-β-gal staining and restored hepatoma cell proliferation. In addition, p53 siRNA transfection attenuated metformin-induced SA-β-gal staining. Intriguingly, co-expression of SIRT1 and p53 dramatically reduced the levels of Ac-p53, however, low doses of metformin treatment partially reversed the effect of SIRT1 on p53 acetylation and elevated SA-β-gal activity. These observations indicate that activation of the AMPK pathway promotes senescence in hepatoma cells exposed to low concentrations of metformin in a p53-dependent manner. Further, low doses of metformin may have the potential to be used as an adjuvant to HCC therapy.

SET-mediated NDRG1 inhibition is involved in acquisition of epithelial-to-mesenchymal transition phenotype and cisplatin resistance in human lung cancer cell

Development of resistance to therapy continues to be a serious clinical problem in lung cancer management. Cancer cells undergoing epithelial-to-mesenchymal transition (EMT) have been shown to play roles in resistance to chemotherapy. Here, we utilized a proteomics-based method and identified a significant downregulation of the metastasis suppressor NDRG1 in drug resistant lung cancer cells. We showed that downregulation of DNRG1 constitutes a mechanism for acquisition of EMT phenotype and endows lung cancer cells with an increased resistance to cisplatin. We also identified a signal cascade, namely, SET--| PP2A--| c-myc--| NDRG1, in which upregulation of SET is critical for inhibition of NDRG1. We also found that blockade of SET (or reactivation of PP2A) by FTY720 reverted EMT, restored drug sensitivity, and inhibited invasiveness and growth of lung tumor xenografts. Together, our results indicated a functional link between SET-mediated NDRG1 regulation and acquisition of EMT phenotype and drug resistance, and provided an evidence that blockade of SET-driven EMT can overcome drug resistance and inhibit tumor progression.

ZEB1 transcriptionally regulated carbonic anhydrase 9 mediates the chemoresistance of tongue cancer via maintaining intracellular pH

BackgroundChemoresistance is a major obstacle in successfully treating cancers, and the mechanisms responsible for drug resistance are still far from understood. Carbonic anhydrase 9 (CA9) has been shown to be upregulated in the drug-resistant tongue cancer cell line Tca8113/PYM and to be associated with drug resistance. However, the mechanisms regulating CA9 expression and its role in drug resistance remain unclear.MethodsBioinformatic and experimental analysis involving ChIP and luciferase reporter assays were used to validate Zinc finger E-box-binding homeobox 1 (ZEB1) as a transcriptional regulator of CA9. Gene expression and protein levels were evaluated by quantitative RT-PCR and western blotting, respectively. Sensitivity to chemotherapy was examined using the MTS assay and Hoechst staining and analysis caspase-3 activity to evaluate changes in apoptosis. Intracellular pH (pHi) was measured using fluorescent pH-indicator BCECF-AM. Protein expression in patient tissue samples was examined by immunohistochemistry and survival of tongue cancer patients from which these samples were derived was also analyzed.ResultsZEB1 bound to the promoter of CA9 to positively regulate CA9 expression in tongue cancer cells. Knockdown of CA9 using short interfering RNA (siRNA) abolished the chemoresistance resulting from ZEB1 overexpression in Tca8113 and SCC-25 cells, and CA9 overexpression attenuated chemosensitivity induced by ZEB1 knockdown in Tca8113/PYM cells. CA9 knockdown also prevented maintenance of pHi mediated by overexpression of ZEB1 in Tca8113 and SCC-25 cells following chemotherapy, associated with increased apoptosis and caspase-3 activation. Conversely, ectopic expression of CA9 suppressed decrease in pHi mediated by ZEB1 knockdown in Tca8113/PYM cells following chemotherapy, accompanied by decreased apoptosis and caspase-3 activation. Importantly, a positive correlation was observed between ZEB1 and CA9 protein expression in tongue cancer tissues, and expression of these proteins associated with a poor prognosis for patients.ConclusionOur finding that tumor cells regulate pHi in response to chemotherapy provides new insights into mechanisms of drug resistance during cancer treatment. Identification of the ZEB1–CA9 signaling axis as a biomarker of poor prognosis in tongue cancer will be valuable in future development of therapeutic strategies aimed at improving treatment efficacy, especially in terms of drug resistance associated with this disease.

miR-493 mediated DKK1 down-regulation confers proliferation, invasion and chemo-resistance in gastric cancer cells

In the present study, we demonstrated that the levels of DKK1 were decreased in serums and tissues of GC. DKK1 levels inversely correlated with tumor class, TNM stage, distant metastasis and lymph node metastasis of GC. GC patients with low DKK1 levels had a poor overall survival. DKK1 inhibited the proliferation of GC cells in vitro and in vivo. DKK1 also inhibited invasion, but enhanced chemo-sensitivity of GC cells. Mechanically, miR-493 levels increased in GC and directly targeted and down-regulated DKK1 expression. In agreement, miR-493 promoted proliferation of GC cells in vitro and in vivo. MiR-493 also promoted invasion and chemo-resistance of GC cells. However, DKK1 overexpression reversed the effects of miR-493 on proliferation, invasion and chemo-sensitivity. Thus, our results provide new insight for the role of miR-493/DKK1 axis in GC.

Cip2a promotes cell cycle progression in triple-negative breast cancer cells by regulating the expression and nuclear export of p27Kip1.

Triple-negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets; as a consequence, TNBC exhibits poor clinical outcome. In this study, we showed that cancerous inhibitor of protein phosphatase 2A (Cip2a) represents a promising target in TNBC because Cip2a was highly expressed in TNBC cells and tumor tissues, and its expression showed an inverse correlation with overall survival in patients with TNBC. We found that inhibition of Cip2a in TNBC cells induced cell cycle arrest at the G2/M phase, inhibited cell proliferation and delayed tumor growth in the xenograft model. Moreover, Cip2a markedly decreased the expression and nuclear localization of p27Kip1 and this is critical for the ability of Cip2a to promote TNBC progression. Mechanistically, our studies showed that Cip2a promoted p27Kip1 phosphoration at Ser10 via inhibiting Akt-associated PP2A activity, which seems to relocalize p27Kip1 to the cytoplasm in TNBC cells. On the other hand, Cip2a also recruited c-myc to mediate the transcriptional inhibition of p27Kip1. Notably, we observed negative correlation between Cip2a and p27Kip1 expression in TNBC specimens. In addition, our data showed that Cip2a depletion could sensitize TNBC to PARP inhibition. Collectively, these data suggested that Cip2a effectively promotes TNBC cell cycle progression and tumor growth via regulation of PP2A/c-myc/p27Kip1 signaling, which could serve as a potential therapeutic target for TNBC patients.

MYCN-mediated miR-21 overexpression enhances chemo-resistance via targeting CADM1 in tongue cancer

Chemo-resistance is still a major obstacle in successful cancer treatment. Previously, we found that miR-21 (miR-21-5p) was upregulated in drug-resistant tongue cancer (TC) cell line Tca8113/PYM. However, the mechanisms for miR-21 upregulation and its role in chemo-resistance in TC remain unclear. Here, we demonstrated that functional inhibition of miR-21 sensitized TC cells to chemotherapy. In agreement, overexpressed miR-21 enhanced chemo-resistance in TC cells. We found that miR-21 directly targeted CADM1 expression, which was downregulated in drug-resistant TC cells. Restored CADM1 expression sensitized TC cells to chemotherapy, but CADM1 knockdown induced chemo-resistance. Mechanically, CADM1 interacted with BMI1 to inhibit its nuclear translocation. Moreover, MYCN which was overexpressed in drug-resistant TC cells directly bound to the miR-21 promoter to upregulated miR-21 expression in TC cells. Importantly, the expression levels of miR-21 and CADM1 negatively correlated, but MYCN and miR-21 positively correlated in TC tissues. High levels of miR-21 and MYCN and low level of CADM1 were associated with poor prognosis in TC patients. In conclusion, our study suggests an important role of the MYCN/miR-21/CADM1 axis in chemo-resistance in TC patients and may lead to promising prognostic biomarkers and novel treatment strategies to improve the chemotherapeutic efficacy for TC patients.Key messagesMiR-21 enhances chemo-resistance via targeting CADM1 in tongue cancer cells.CADM1 sensitizes tongue cancer cells to chemotherapy.CADM1 interacts with BMI1 to inhibit its nuclear translocation.MYCN transcriptionally regulates miR-21 expression.Dysregulated MYCN/miR-21/CADM1 axis associates with poor prognosis in TC patients.

HSP27-Mediated Extracellular and Intracellular Signaling Pathways Synergistically Confer Chemoresistance in Squamous Cell Carcinoma of Tongue

Purpose: Squamous cell carcinoma of tongue (SCCT) is the most common type of oral cavity carcinoma. Chemoresistance in SCCT is common, and the underlying mechanism remains largely unknown. We aimed to identify key molecules and signaling pathways mediating chemoresistance in SCCT. Experimental Design: Using a proteomic approach, we found that the HSP27 was a potential mediator for chemoresistance in SCCT cells. To further validate this role of HSP27, we performed various mechanistic studies using in vitro and in vivo models as well as serum and tissue samples from SCCT patients. Results: The HSP27 protein level was significantly increased in the multidrug-resistant SCCT cells and cell culture medium. Both HSP27 knockdown and anti-HSP27 antibody treatment reversed chemoresistance. Inversely, both HSP27 overexpression and recombinant human HSP27 protein treatment enhanced chemoresistance. Moreover, chemotherapy significantly induced HSP27 protein expression in both SCCT cells and their culture medium, as well as in tumor tissues and serum of SCCT patients. HSP27 overexpression predicts a poor outcome for SCCT patients receiving chemotherapy. Mechanically, extracellular HSP27 binds to TLR5 and then activates NF-κB signaling to maintain SCCT cell survival. TLR5 knockdown or restored IκBα protein level disrupts extracellular HSP27-induced NF-κB transactivation and chemoresistance. Moreover, intracellular HSP27 binds to BAX and BIM to repress their translocation to mitochondrion and subsequent cytochrome C release upon chemotherapy, resulting in inhibition of the mitochondrial apoptotic pathway. Conclusions: HSP27 plays a pivotal role in chemoresistance of SCCT cells via a synergistic extracellular and intracellular signaling. HSP27 may represent a potential biomarker and therapeutic target for precision SCCT treatment. Clin Cancer Res; 24(5); 1163–75. ©2017 AACR.

LINC01638 lncRNA activates MTDH-Twist1 signaling by preventing SPOP-mediated c-Myc degradation in triple-negative breast cancer

Breast cancer is a heterogeneous disease, and triple-negative breast cancer (TNBC) continues to be a serious health problem. The potential involvement of lncRNAs in TNBC progression remains unexplored. Here, we demonstrated that LINC01638 is highly expressed in TNBC tissues and cells. LINC01638 maintains the mesenchymal traits of TNBC cells, including an enriched epithelial-mesenchymal transition (EMT) signature and cancer stem cell-like state. LINC01638 knockdown suppresses tumor proliferation and metastasis both in vitro and in vivo. LINC01638 overexpression predicts a poor outcome of breast cancer patients. Mechanistically, LINC01638 interacts with c-Myc to prevent SPOP-mediated c-Myc ubiquitination and degradation. C-Myc transcriptionally enhances MTDH (metadherin) expression and subsequently activates Twist1 expression to induce EMT. Our findings describe LINC01638-mediated signal transduction and highlight the crucial role of LINC01638 in TNBC progression.

Simultaneous amplification of exons 18 to 21 of the EGFR gene using 5′ tailed primers and a two-stage protocol

ABSTRACT Objective: Reduction of non-specific amplification and achievement of efficient amplification of multiple gene fragments under the same reaction condition is the basic goal of PCR diagnosis; however, this is often difficult. This study was conducted to establish a highly specific and effective amplification of the epidermal growth factor receptor (EGFR) genes exons, 18–21, simultaneously. Methods: The 5′-tailed primers were synthesized by adding 10 to 20 bp of a non-specific sequence to the 5′-terminus of sequence-specific primers (tailless primers). The two-stage protocol consisted of 5–10 cycles of a conventional 3-step cycling, which was then followed by 30–35 cycles of two-step cycling. The exons 18–21 of EGFR gene were amplified in 28 non-small cell lung cancer (NSCLC) patients using an optimized PCR that combined 5′ tailed primers with a two-stage protocol. Results: The 5′ tailed primers exhibited a wider range of suitable annealing temperatures, similar range of primer concentration, similar sensitivity, specificity, and reproducibility, as well as a reduced, non-specific amplification compared with the corresponding tailless primers. The amplification of exons 18–21 of EGFR gene in NSCLC patients revealed that a combination of 5′ tailed primers with two-stage protocol (optimized PCR) had a similar PCR success rate (P = 0.873) but had significantly reduced non-specific amplification (P <0.001) compared to conventional PCR. Conclusion: 5′ tailed primers exhibited a wider range of suitable annealing temperatures and improved specificity compared with conventional PCR primers. An optimized PCR was established with 5′ tailed primers and a two-stage protocol to amplify exons 18–21 of the EGFR gene in NSCLC patients.

Activation of FOXO3a reverses 5-Fluorouracil resistance in human breast cancer cells

Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide. Drug resistance is the major obstacle for breast cancer treatment improvement. TRAIL-inducing compound 10 (Tic10), a novel activator of FOXO3, exhibits potent antitumor efficacy both in vitro and in vivo. In the present study, we investigated the resistance reversal effect of Tic10 on multidrug-resistant breast cancer cells T47D/5Fu derived from T47D breast cancer cells. We found that FOXO3a was significantly decreased in T47D/5-Fu cells, whereas treatment of Tic10 enhances FOXO3a expression and nuclear translocation. Moreover, treatment of Tic10 could reverses 5-Fluorouracil resistance of T47D/5-Fu cells via induction of G0/G1 cell cycle arrest and apoptosis. Furthermore, we found that Tic10 decreased the expression of CDK4 via FOXO3a-dependment mechanism. In addition, our data showed that Tic10 could sensitize drug resistant T47D/5-Fu cells to 5-Fu in vivo. Taken together, these data suggested Tic10 as capable of restoring sensitivity for drug-resistant breast cancer cells.