A novel and feasible strategy to specifically inhibit PKCα expression and tumor development

Breast cancer is the leading cause of women malignancy in the world with an increasing trend in recent decades whether in Western country or Asia. The modern westernized life style with high fat food accompany with the high prevalence. Several types of breast cancer had been classified upon pathological feature according to expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her-2). However, the triple negative breast cancer had no expression of above receptors which made it more difficult in treatment since there was no space for hormone and target therapy in treating patient. Therefore the triple negative breast cancer had easy recurrence and metastasis with poor outcome. Recent reports demonstrated that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) is correlated with poorer survival outcomes. However, the mechanism of the enhanced expression of PKCα in TNBC remains greatly unknown. In the present study, our results showed that the presence of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) expressions correlate to PKCα expression in TNBC. Furthermore, it is the interaction between the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 which facilitated their heterodimeric complex formation before their binding to the PKCα promoter. Blocking the formation of the heterodimer changed Elk-1 nuclear localization, MZF-1 protein degradation, their DNA-binding activities, and subsequently the expression of PKCα in TNBC cells. Thus, migration, tumorigenicity, and epithelial–mesenchymal transition potential of TNBC cells decreased, suggesting that the Elk-1/MZF-1 heterodimer is considered as a mediator of PKCα in TNBC cell malignancy. The obtained data also suggested that the next therapeutic strategy in the treatment of TNBC will come from the blocking of Elk-1/MZF-1 interaction through the saturation of Elk-1 or MZF-1 binding domains, such as through the application of cell-penetrating HIV transactivating regulatory protein-fused peptides to inhibit tumor growth and metastasis.乳癌是女性癌症中最常見的惡性腫瘤，近十年來，無論是在西方國家還是亞洲，其發生率都有逐年增加的趨勢。尤其是高脂的飲食習慣，更會提高乳癌的發率。乳癌的分類，可依據其病理化驗中，雌激素受體 (estrogen receptor；ER)、黃體素受體 (progesterone receptor；PR) 和人類上皮細胞生長因子接受體 2 (human epidermal growth factor receptor 2；Her-2) 分別的表現量來進行分類。但是，在三陰性乳癌的患者中，上述的三種受體都沒有表現，使得賀爾蒙治療和抗HER2標靶治療藥物都不適用，進而增加了治療的難度。因此，三陰性乳癌較其他乳癌具有較高的復發機率、轉移到其他器官的風險和較差的預後。近年來的研究結果證實，在三陰性乳癌，其蛋白激酶C α (protein kinase C alpha；PKCα) 的表現量與病人的存活率有著負相關的情形。然而，是什麼機制使得PKCα大量的表現在三陰性乳癌中，目前仍然是未知。本研究中，我們發現，在三陰性乳癌中MZF-1 (myeloid zinc finger 1) 和Elk-1 (Ets-like protein-1) 的表現量與PKCα有正相關的情形。此外，在PKCα被活化之前，MZF-1的酸性結合區域 (acidic domain) 與Elk-1的肝素結合區域 (heparin-binding domain) 能相互結合，形成二聚體。當MZF-1發生蛋白質降解，阻斷此二具體的形成，會使得Elk-1進入細胞核內，進而結合在PKCα的啟動子 (promoter) 上，促進PKCα的產生，進而促進了腫瘤細胞的轉移、癌化、上皮細胞中胚轉化 (epithelial-mesenchymal transition；EMT) 的現象，因此推測，Elk-1/MZF-1 二具體在三陰性乳癌中，是一個很重要的PKCα調節因子。由我們的實驗結果也證實，透過人類免疫缺損病毒HIV-1內的TAT蛋白衍生物 (TAT 細胞穿膜肽) 的應用，來阻斷Elk-1/MZF-1 二具體的形成，能有效的抑制腫瘤的生長和轉移，這或許能成為治療三陰性乳癌的另一個新的治療策略。Acknowledgements i 中文摘要 ii Abstract iii Figure contents viii Table content xiii 1. Background 1 1.1 Epidemiology of Breast Cancer 1 1.2 Classification of Breast Cancer 1 1.3 Triple-negative Breast Cancer 2 1.4 Triple-negative Breast Cancer and Protein Kinase C alpha (PKCα) 3 1.5 Hypothesis 6 1.6 Goals and Aims 7 2. Experimental Design 8 2.1 Immunohistochemical (IHC) Staining 8 2.2 Plasmid Construction 9 2.3 Cell Lines 11 2.4 Luciferase Reporter Assay 12 2.5 Electrophoretic Mobility Shift Assay 14 2.6 Co-Immunoprecipitation and Immunoblotting Analysis 15 2.7 Chromatin Immunoprecipitation (ChIP) Assay 16 2.8 Antisense Knockout Assays 17 2.9 Cell Proliferation, Migration, and Invasion Assays 17 2.10 Animal Studies 18 2.11 Confocal Immunofluorescence Microscopy 19 2.12 Microarray Analysis 19 2.13 Statistical Analysis 20 3. Results 21 3.1 Expression of PKCα Correlates with MZF-1/Elk-1 in TNBC 21 3.2 MZF-1/Elk-1 Complex Binds to the Promoter Region of PRKCA 21 3.3 The Acidic Domain of MZF-1 Interacts with the Heparin-Binding Domain of Elk-1 23 3.4 Inhibition of MZF-1 and Elk-1 Heterodimer Formation Attenuates Drug Resistance and Malignant Phenotypes of Breast Cancer Cells by Reducing PKCα Expression 25 3.5 Blocking MZF-1/Elk-1 Heterodimer Formation Decreases EMT Potential 27 3.6 TAT-Fused Peptides Inhibit MZF-1/Elk-1 Heterodimer Formation 29 4. Discussion 31 4.1 Elk-1/MZF-1 Heterodimers Regulate PKCα Expression and their Functions in TNBC 31 4.2 Elk-1/MZF-1 Heterodimers Promote EMT 33 5. Summary 34 6. Figures 36 7 Table 60 8. Supplemental Figures 62 9. References 116 10. Appendix 12

MZF-1/Elk-1 Complex Binds to Protein Kinase Cα Promoter and Is Involved in Hepatocellular Carcinoma

In this study, the molecular mechanism of protein kinase C alpha (PKCα) gene regulation in hepatocellular carcinoma (HCC) involving Ets-like protein-1 (Elk-1) and myeloid zinc finger-1 (MZF-1) was investigated. The luciferase reporter assay results revealed that the presence of both MZF-1 and Elk-1 significantly contributed to the upregulation of PKCα gene transcription activity, and the transcriptional activity decreased when the transfection included a DNA-binding-deficient (?DBD) gene vector of either MZF-1 or Elk-1 DNA-binding deficiency (MZF-1?DBD or Elk-1?DBD), thereby indicating that the enhanced expression of PKCα was caused by the binding of MZF-1 and/or Elk-1 with the PKCα promoter. We investigated MZF-1 and Elk-1 to determine whether they bind to each other. The results of immunoprecipitation (IP), Co-IP, chromatin IP (ChIP), and Re-ChIP analyses indicated that Elk-1 can directly bind to the N-terminal region of MZF-1 and MZF-1 can directly bind to the C-terminal region of Elk-1 to form a complex before attaching to the PKCα promoter. Furthermore, when MZF-1?DBD or Elk-1?DBD was added to the cells, PKCα expression decreased, and cell proliferation, migration, invasion, and tumorigenicity also decreased. These findings suggest that PKCα expression in HCC could be stimulated by the formation of MZF-1/Elk-1 complex, which directly binds to the PKCα promoter

Emodin and Aloe-Emodin Suppress Breast Cancer Cell Proliferation through ERα Inhibition

The anthraquinones emodin and aloe-emodin are abundant in rhubarb. Several lines of evidence indicate that emodin and aloe-emodin have estrogenic activity as phytoestrogens. However, their effects on estrogen receptor α (ERα) activation and breast cancer cell growth remain controversial. The goal of this study is to investigate the effects and molecular mechanisms of emodin and aloe-emodin on breast cancer cell proliferation. Our results indicate that both emodin and aloe-emodin are capable of inhibiting breast cancer cell proliferation by downregulating ERα protein levels, thereby suppressing ERα transcriptional activation. Furthermore, aloe-emodin treatment led to the dissociation of heat shock protein 90 (HSP90) and ERα and increased ERα ubiquitination. Although emodin had similar effects to aloe-emodin, it was not capable of promoting HSP90/ERα dissociation and ERα ubiquitination. Protein fractionation results suggest that aloe-emodin tended to induce cytosolic ERα degradation. Although emodin might induce cytosolic ERα degradation, it primarily affected nuclear ERα distribution similar to the action of estrogen when protein degradation was blocked. In conclusion, our data demonstrate that emodin and aloe-emodin specifically suppress breast cancer cell proliferation by targeting ERα protein stability through distinct mechanisms. These findings suggest a possible application of anthraquinones in preventing or treating breast cancer in the future

MZF-1/Elk-1 Complex Binds to Protein Kinase Cα Promoter and Is Involved in Hepatocellular Carcinoma.

In this study, the molecular mechanism of protein kinase C alpha (PKCα) gene regulation in hepatocellular carcinoma (HCC) involving Ets-like protein-1 (Elk-1) and myeloid zinc finger-1 (MZF-1) was investigated. The luciferase reporter assay results revealed that the presence of both MZF-1 and Elk-1 significantly contributed to the upregulation of PKCα gene transcription activity, and the transcriptional activity decreased when the transfection included a DNA-binding-deficient (∆DBD) gene vector of either MZF-1 or Elk-1 DNA-binding deficiency (MZF-1∆DBD or Elk-1∆DBD), thereby indicating that the enhanced expression of PKCα was caused by the binding of MZF-1 and/or Elk-1 with the PKCα promoter. We investigated MZF-1 and Elk-1 to determine whether they bind to each other. The results of immunoprecipitation (IP), Co-IP, chromatin IP (ChIP), and Re-ChIP analyses indicated that Elk-1 can directly bind to the N-terminal region of MZF-1 and MZF-1 can directly bind to the C-terminal region of Elk-1 to form a complex before attaching to the PKCα promoter. Furthermore, when MZF-1∆DBD or Elk-1∆DBD was added to the cells, PKCα expression decreased, and cell proliferation, migration, invasion, and tumorigenicity also decreased. These findings suggest that PKCα expression in HCC could be stimulated by the formation of MZF-1/Elk-1 complex, which directly binds to the PKCα promoter

Suppression of breast cancer cell growth by Her2-reduced AR serine 81 phosphorylation

Breast cancer is a hormone-related carcinoma and the most commonly diagnosed malignancy in women. Although Her-2, estrogen receptor (ER), and progesterone receptor (PR) are the major diagnostic markers and therapeutic targets to breast cancer, searching for additional molecular targets remains an important issue and one of the candidates is androgen receptor (AR). AR has been shown expressed in 70% breast cancer patients and connects to low recurrence and high survival rate. Our previous study demonstrates that Ser81 phosphorylation of AR in prostate cancer cells is critical for its protein stability modulated by human epidermal growth factor receptor-2 (Her2). The aim of this study is to investigate the influence of Her2 and AR in proliferation of breast cancer cell line, MDA-MB-453. The data show that AR which was activated by synthetic androgen R1881 suppressed the proliferation of MDA-MB-453 cells. Notably, AR activation decreased the protein levels of cell growth-related proteins, including cyclin A, cyclin B, and early growth response protein 1 (Egr1), while cell-cycle inhibitor protein p27 was increased. Besides, Heregulin (HRG)-induced Her2 activation decreased the AR protein levels and its Ser81 phosphorylation. Her2 small molecular inhibitor, Lapatinib, dose-dependently suppressed cell proliferation while the levels of phospho-Ser81 AR and p27 protein were increased. Phospho-Ser81 AR was also increased after Her2 knockdown. Specifically, the influence of phospho-Ser81 AR by Lapatinib was primarily found in the nucleus of MDA-MD-453 cells, where the cell proliferation might directly be interfered. In conclusion, our findings indicate that Her2 might negatively regulate AR phosphorylation/activation and contribute to regulate the proliferation of MDA-MB 453 cells

RET Regulates Human Medullary Thyroid Cancer Cell Proliferation through CDK5 and STAT3 Activation

Medullary thyroid cancer (MTC) is a neuroendocrine tumor that arises from the parafollicular C-cells, which produces the hormone calcitonin. RET is a transmembrane receptor protein-tyrosine kinase, which is highly expressed in MTC. Our previous studies reported that cyclin-dependent kinase 5 (CDK5) plays a crucial role in cancer progression, including MTC. However, the role of CDK5 in GDNF-induced RET signaling in medullary thyroid cancer proliferation remains unknown. Here, we investigated RET activation and its biochemically interaction with CDK5 in GDNF-induced medullary thyroid cancer proliferation. Our results demonstrated that GDNF stimulated RET phosphorylation and thus subsequently resulted in CDK5 activation by its phosphorylation. Activated CDK5 further caused STAT3 activation by its specific phosphorylation at Ser727. Moreover, we also found that GDNF treatment enhanced ERK1/2 and EGR1 activity, which is involved in p35 activation. Interestingly, we identified for the first time that CDK5 physically interacted with RET protein in MTC. Overall, our results provide a new mechanism for medullary thyroid cancer cell proliferation, suggesting that targeting CDK5 may be a promising therapeutic candidate for human medullary thyroid cancer in the near future

Suppression of tumorigenesis in MZF-1ΔDBD-transfected cells.

<p>(A) Tumor growth curve in FLAG (5 μg; ■) or MZF-1ΔDBD (5 μg;●) transfected human SK-Hep-1 xenografts (n = 5). A total of 5 × 10⁷ FLAG- or MZF-1ΔDBD-pretreated cells were injected into the flank region of nude mice. Tumor development was observed in 2 months, and the mice were then sacrificed. Cross-sectional tumor diameters were measured externally, and the approximate tumor volume was calculated as described in the Materials and Methods Section. The results represent the mean ± SE of five developed tumors in the experimental mice. **<i>P</i> < 0.01 vs. FLAG pretreated group. (B) Visible tumor (white arrow) formed by the indicated pretreated cells on day 56. The blots of the anti-FLAG indicated the presence of exogenous MZF-1-(1–72) in cells. β-actin was designed as a housekeeping gene.</p