Jiao Meng

Aurora-A controls cancer cell radio- and chemoresistance via ATM/Chk2-mediated DNA repair networks

High expression of Aurora kinase A (Aurora-A) has been found to confer cancer cell radio- and chemoresistance, however, the underlying mechanism is unclear. In this study, by using Aurora-A cDNA/shRNA or the specific inhibitor VX680, we show that Aurora-A upregulates cell proliferation, cell cycle progression, and anchorage-independent growth to enhance cell resistance to cisplatin and X-ray irradiation through dysregulation of DNA damage repair networks. Mechanistic studies showed that Aurora-A promoted the expression of ATM/Chk2, but suppressed the expression of BRCA1/2, ATR/Chk1, p53, pp53 (Ser15), H2AX, γH2AX (Ser319), and RAD51. Aurora-A inhibited the focus formation of γH2AX in response to ionizing irradiation. Treatment of cells overexpressing Aurora-A and ATM/Chk2 with the ATM specific inhibitor KU-55933 increased the cell sensitivity to cisplatin and irradiation through increasing the phosphorylation of p53 at Ser15 and inhibiting the expression of Chk2, γH2AX (Ser319), and RAD51. Further study revealed that BRCA1/2 counteracted the function of Aurora-A to suppress the expression of ATM/Chk2, but to activate the expression of ATR/Chk1, pp53, γH2AX, and RAD51, leading to the enhanced cell sensitivity to irradiation and cisplatin, which was also supported by the results from animal assays. Thus, our data provide strong evidences that Aurora-A and BRCA1/2 inversely control the sensitivity of cancer cells to radio- and chemotherapy through the ATM/Chk2-mediated DNA repair networks, indicating that the DNA repair molecules including ATM/Chk2 may be considered for the targeted therapy against cancers with overexpression of Aurora-A.

Small Ribosomal Protein Subunit S7 Suppresses Ovarian Tumorigenesis through Regulation of the PI3K/AKT and MAPK Pathways

Small ribosomal protein subunit S7 (RPS7) has been reported to be associated with various malignancies, but the role of RPS7 in ovarian cancer remains unclear. In this study, we found that silencing of RPS7 by a specific shRNA promoted ovarian cancer cell proliferation, accelerated cell cycle progression, and slightly reduced cell apoptosis and response to cisplatin treatment. Knockdown of RPS7 resulted in increased expression of P85α, P110α, and AKT2. Although the basal levels of ERK1/2, MEK1/2, and P38 were inconsistently altered in ovarian cancer cells, the phosphorylated forms of MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), JNK1/2 (Thr183/Tyr185), and P38 (Thr180/Tyr182) were consistently reduced after RPS7 was silenced. Both the in vitro anchorage-independent colony formation and in vivo animal tumor formation capability of cells were enhanced after RPS7 was depleted. We also showed that silencing of RPS7 enhanced ovarian cancer cell migration and invasion. In sum, our results suggest that RPS7 suppresses ovarian tumorigenesis and metastasis through PI3K/AKT and MAPK signal pathways. Thus, RPS7 may be used as a potential marker for diagnosis and treatment of ovarian cancer.

Stanniocalicin 2 Suppresses Breast Cancer Cell Migration and Invasion via the PKC/Claudin-1-Mediated Signaling

Stanniocalcin (STC), a glycoprotein hormone, is expressed in a wide variety of tissues to regulate Ca2+ and PO4- homeostasis. STC2, a member of STC family, has been reported to be associated with tumor development. In this study, we investigated whether the expression of STC2 is associated with migration and invasion of breast cancer cells. We found that breast cancer cell line 231 HM transfected with STC2 shRNA displayed high motility, fibroblast morphology, and enhanced cell migration and invasion. Introduction of STC2 in 231 cells reduced cell migration and invasion. In response to irradiation, silencing of STC2 in 231 HM cells reduced apoptosis, whereas overexpression of STC2 in 231 cells promoted apoptosis, compared with in control cells. Mechanistic study showed that STC2 negatively regulated PKC to control the expression of Claudin-1, which subsequently induced the expressions of EMT-related factors including ZEB1, ZO-1, Slug, Twist, and MMP9. Suppression of PKC activity by using a PKC inhibitor (Go 6983) restored the normal motility of STC2-silenced cells. Furthermore, in vivo animal assay showed that STC2 inhibited tumorigenesis and metastasis of breast cancer cells. Collectively, these results indicate that STC2 may inhibit EMT at least partially through the PKC/Claudin-1-mediated signaling in human breast cancer cells. Thus, STC2 may be exploited as a biomarker for metastasis and targeted therapy in human breast cancer.

COX-2 promotes breast cancer cell radioresistance via p38/MAPK-mediated cellular anti-apoptosis and invasiveness

Radioresistance is one of the major barriers to improve the survival rate of breast cancer patients. Cyclooxygenase 2 (COX-2) is usually overexpressed in highly invasive and metastatic breast cancer, which may indicate an association with breast cancer radioresistance. The function role of COX-2 was investigated by using a radioresistant breast cancer cell line MDA-MB-231/RR10 and its parental cell line MDA-MB-231 cells before or after COX-2 was silenced by a specific small hairpin RNA (shRNA). The cell proliferation, migration, invasion, colony formation, and apoptosis were measured by CCK-8, scratch-wound, transwell, clone formation assay, and flow cytometry. Protein and mRNA expression were analyzed by Western blot and quantitative reverse transcriptase-polymerase chain reaction. COX-2 is upregulated in MDA-MB-231/RR10 cells compared with in MDA-MB-231 cells, and silencing of COX-2 expression by shRNA in MDA-MB-231/RR10 cells decreases the expression of Bcl-2 and Bcl-XL, but increases the proapoptotic protein BAK, leading to the increased apoptosis following treatment with γ-irradiation in comparison with those in control cells. Silencing of COX-2 also increases the expression of β-catenin and E-cadherin, two anti-invasion proteins, resulting in reduced cell migration and invasion tested by transwell chambers and wound-healing assays. Further study demonstrated that COX-2-induced radioresistance is negatively regulated through the phosphorylation of p38 at Tyr182, and that the phosphorylation of p38 induced by TNF-alpha reduces the expression of Bcl-2, BCL-XL, but increases β-catenin and E-cadherin, leading to the decreased invasiveness of cells. Our data suggest that COX-2, p38, Bcl-2, Bcl-XL, β-catenin, and E-cadherin may be considered as potential therapeutic targets against radioresistant breast cancer.

Aurora-A: a potential DNA repair modulator

It is well-known that overexpression of Aurora-A promotes tumorigenesis, but the role of Aurora-A in the development of cancer has not been fully investigated. Recent studies indicate that Aurora-A may confer cancer cell chemo- and radioresistance through dysregulation of cell cycle progression and DNA damage response. Direct evidences from literatures suggest that Aurora-A inhibits pRb, p53, p21waf1/cip1, and p27cip/kip but enhances Plk1, CDC25, CDK1, and cyclin B1 to repeal cell cycle checkpoints and to promote cell cycle progression. Other studies indicate that Aurora-A suppresses BRCA1, BRCA2, RAD51, poly(ADP ribose) polymerase (PARP), and gamma-H2AX to dysregulate DNA damage response. Aurora-A may also interact with RAS and Myc to control DNA repair indirectly. In this review, we summarized the potential role of Aurora-A in DNA repair from the current literatures and concluded that Aurora-A may function as a DNA repair modulator to control cancer cell radio- and chemosensitivity, and that Aurora-A-associated DNA repair molecules may be considered for targeted cancer therapy.

The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy

It is well known that the activation of Aurora A/B (Aur A/B) or inactivation of BRCA1/2 induces tumor formation. Others and we have reported that the mutual suppression between Aur A/B and BRCA1/2 may manipulate cancer cell growth and tumorigenesis, however, the interactive regulation and mechanism between these molecules are still elusive. In this study, by consecutive silencing of Aur A/B or/and BRCA1/2 with specific shRNAs, we showed that, in BRCA2-deficient pancreatic cancer cell line Capan-1 and in ovarian cancer cell line OVCA433, Aur A/B and BRCA1/2 inversely regulated the expression of each other likely through proteasome-mediated proteolysis but not through gene transcription. Aur A/B and BRCA1/2 conversely regulated cell cycle progression mainly through control of p53 and cyclin A. Moreover, the disruption of Aur A/B blocked abnormal cytokinesis and decreased cell multinuclearity and chromosome tetraploidy, whereas the deprivation of BRCA1/2 promoted the abnormal cytokinesis and enhanced the cell multinuclearity and tetraploidy. Furthermore, we showed by animal assays that the depletion of Aur A/B inhibited tumor growth of both cell lines, while the knockdown of BRCA1/2 promoted the tumor growth. However, the concurrent silencing of Aur A/B and BRCA1/2 diminished the effects of these molecules on the regulation of cell cycle, cytokinesis, and tetraploidy, leading to the burdened tumor sizes similar to those induced by scrambled shRNA-treated control cells. In summary, our study revealed that the negative interplay between Aur A/B and BRCA1/2 inversely controls the cell proliferation, cell cycle progression, cell multinuclearity, and tetraploidization to modulate tumorigenesis.

CXCR2 promotes breast cancer metastasis and chemoresistance via suppression of AKT1 and activation of COX2

Metastasis and chemoresistance are two major causes of breast cancer death. We show here that the chemokine receptor CXCR2 was overexpressed in breast cancer cell lines and tissues. CXCR2 promoted anti-apoptosis, anti-senescence, and epithelial-to-mesenchymal transition (EMT) of breast cancer cells, leading to the enhanced metastasis and chemoresistance. Further study suggested that AKT1 and cyclooxygenase-2 (COX2; PTGS2) might mediate the CXCR2 signaling to inversely control the breast cancer metastasis and chemoresistance through the regulation of EMT, apoptosis, and senescence. Analyses of clinical data indicate that the high expression of CXCR2 was correlated with the high expression of COX2 and the low expression of AKT1, P85α, E-cadherin, and β-catenin in cancer tissues. Poor outcomes were associated with the high expression of CXCR2 or COX2 while favorable survivals were associated with the high expression of P85α, AKT1, or E-cadherin in all cancer patients. Cox multivariate analysis demonstrated that CXCR2, COX2, and AKT1 could be independent predictors for disease free survivals. All these data suggest that CXCR2 promotes breast cancer metastasis and chemoresistance via suppressing AKT1 and activating COX2. Thus, antagonists of the CXCR2 signaling molecules may be used to treat breast cancer patients particularly with high metastasis and chemoresistance.

STC2 promotes head and neck squamous cell carcinoma metastasis through modulating the PI3K/AKT/Snail signaling

The mammalian peptide hormone stanniocalcin 2 (STC2) plays an oncogenic role in many human cancers. However, the exact function of STC2 in human head and neck squamous cell carcinoma (HNSCC) is unclear. We aimed to examine the function and clinical significance of STC2 in HNSCC. Using in vitro and in vivo assays, we show that overexpression of STC2 suppressed cell apoptosis, promoted cell proliferation, migration, invasion, and cell cycle arrest at the G1/S transition. By contrast, silencing of STC2 inhibited these activities. We further show that STC2 upregulated the phosphorylation of AKT and enhanced HNSCC metastasis via Snail-mediated increase of vimentin and decrease of E-cadherin. These responses were blocked by silencing of STC2/Snail expression or inhibition of pAKT activity. Furthermore, clinical data indicate that high STC2 expression was associated with high levels of pAKT and Snail in tumor samples from HNSCC patients with regional lymph node metastasis (P < 0.01). Thus, we conclude that STC2 controls HNSCC metastasis via the PI3K/AKT/Snail signaling axis and that targeted therapy against STC2 may be a novel strategy to effectively treat patients with metastatic HNSCC.

Wip1 suppresses ovarian cancer metastasis through the ATM/AKT/Snail mediated signaling

Inactivation of p53 greatly contributes to serous ovarian cancer, while the role of the wild-type p53 induced phosphatase 1 (Wip1) is quite unclear. In this study, by silencing or overexpression of Wip1, we found that Wip1 suppressed ovarian cancer cell invasion, migration, epithelial to mesenchymal transition (EMT), and ovarian cancer metastasis in xenograft animal models. Mechanistic studies showed that Wip1 may block ovarian cancer metastasis through inhibition of Snail and p-Akt expression because silencing or overexpression of Wip1 either upregulated or downregulated the expression of Snail and p-Akt (Ser 473), while further knockdown of Snail by shRNA or inhibition of p-Akt by a chemical compound attenuated cell invasion, migration and EMT in Wip1 silencing cells. We also found that the phosphorylation of Akt at Ser 473 might be mediated through p-ATM (Ser 1981). Thus, Wip1 may suppress ovarian cancer metastasis through negative regulation of p-ATM, p-Akt, and Snail, which was also evidenced in the limited clinical specimens. Therefore, our data may provide a novel therapeutic indication for serous ovarian cancer based on the uncovered mechanism associated with the precise function of Wip1 independent of p53.

Abstract 1605: Aurora-A promotes radio- and chemo- resistance through regulation of DNA damage response proteins.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Poor prognosis of cancer is usually resulted from radio- and chemo-resistance. Aurora-A, a serine/threonine kinase, associated with spindle separation and centrosome duplication, is overexpressed in many cancers. Purposes: High expression of Aurora-A negatively regulates BRCA2, indicating that Aurora-A is probably associated with DNA damage repair, which may mediate resistance of radio- and chemo-therapy. This study was to investigate the role of Aurora-A in radio- and chemo-resistance in order to seek novel therapeutic methods for human cancer. Materials and methods: Cell lines from breast cancer, pancreatic cancer and ovarian cancer, including those with either low Aurora-A expression (MCF-7, PANC-1 and OVCA420), or high Aurora-A expression (MDA-MB-231, BXPC3 and OVCA429), were used to overexpress or silence Aurora-A, BRCA1, or BRCA2. Cell growth curve and colony formation were tested by cell counting and soft agar assay. Flow cytometry and MTT were used to examine cell apoptosis or cell viability (IC50) after treatment with radiation and cisplatin or VX680 (Aurora-A inhibitor). Western blotting was used to test the expression of DNA damage responsive proteins. Immunofluorescence was used to detect intracellular localization of above proteins. Results: Our data showed that introduction of Aurora-A or silencing of BRCA1 and BRCA2 increased cell growth and colony formation, while silencing of Aurora-A or introduction of BRCA1 and BRCA2 decreased cell proliferation and colony formation. Introduction of Aurora-A also reduced apoptosis in response to gamma-irradiation and cisplatin treatment, while silencing of Aurora-A or treatment of cells with VX680 enhanced apoptosis. Introduction of Aurora-A increased cell viability and IC50 in response to cisplatin treatment, while knockdown of Aurora-A or treatment with VX680 decreased cell viability and IC50. Western blot suggested that Aurora-A had a negative association with BRCA1 and BRCA2. Moreover, overexpression of Aurora-A either by introduction of Aurora-A cDNA or knockdown of BRCA1 or BRCA2 attenuated the expression of DNA damage responsive proteins including RAD51, H2AX, γH2AX, ATR, ERCC2, Chk1, pChk1, Chk2, pChk2,p53, pp53, DNApolζ, which was confirmed by Immunofluorescence. Conclusions: Aurora-A regulates DNA damage responsive proteins including BRCA1, BRCA2, RAD51, H2AX, ERCC2, Chk1, Chk2, ATR, DNA pol ζ to confer cancer radio- and chemo-resistance. Therefore, DNA damage pathway should be essentially considered in the targeted therapy against Aurora-A in cancer patients either with activated Aurora-A or with inactivated BRCA1 or BRCA2. Citation Format: Huizhen Sun, Ziliang Wang, Jiao Meng, Yan Wang, Zihao Qi, Han Xu, Fengjuan Lin, Wen Gao, Jianmin Sun, Jing Hou, Gong Yang. Aurora-A promotes radio- and chemo- resistance through regulation of DNA damage response proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1605. doi:10.1158/1538-7445.AM2013-1605 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.