Sun-Hye Lee

Role of CK1 in GSK3β-mediated phosphorylation and degradation of Snail

The epithelial to mesenchymal transition (EMT) that occurs during embryonic development has begun to attract attention as a potential mechanism for tumor cell metastasis. Snail is a well-known Zn-finger transcription factor that promotes EMT by repressing E-cadherin expression. It is known that Snail is phosphorylated by GSK3β and degraded by β-TrCP-mediated ubiquitination. Here we described another protein kinase, CK1, whose phosphorylation of Snail is required for the subsequent GSK3β phosphorylation. Specific inhibition or depletion of CK1ɛ inhibits the phosphorylation and degradation of Snail and promotes cell migration, suggesting a central role of CK1ɛ in the EMT process. Furthermore, our study uncovered distinct roles and steps of Snail phosphorylation by CK1ɛ and GSK3β. Taken together, we identified CK1ɛ as a new component of the Snail-mediated EMT process, providing insight into the mechanism of human cancer metastasis.

p53, secreted by K-Ras-Snail pathway, is endocytosed by K-Ras-mutated cells; implication of target-specific drug delivery and early diagnostic marker.

p53 is eliminated from K-Ras-mutated cancer cells through direct interaction with Snail. However, it is not achieved through proteasome-mediated degradation or transcriptional repression. Here we provide evidence that p53, binding with Snail, is exported from a K-Ras-mutated cell through a vesicle transport-like mechanism, independently using a p53-nuclear-exporting mechanism. Although we can detect p53 in culture media, a majority of p53 might be degraded by extracellular proteases. Thus, we can recover the secreted p53 in culture media by the inhibition of protease and endocytosis. In addition, a considerable amount of p53 is endocytosed by neighboring cells. As p53 resorption occurs in a K-Ras-dependent manner, treatment of recombinant p53 is detected in the whole-cell lysate of K-Ras-mutated cells, but not in that of wild-type cells. Using the property of p53, we can deliver the chemical (propidium iodine) into K-Ras mutated cells selectively. In contrast, Snail, a co-secreted protein with p53 in response to oncogenic K-Ras, shows resistance to endocytosis and protease, and results in remaining in the media. Thus, we can detect an autoantibody against Snail in the serum of a human cancer patient. Our finding can be used for a mutant K-Ras-specific anticancer drug delivery system and for the diagnosis of pancreatic, colon and lung cancers.

Novel tumor suppressive function of Smad4 in serum starvation-induced cell death through PAK1–PUMA pathway

DPC4 (deleted in pancreatic cancer 4)/Smad4 is an essential factor in transforming growth factor (TGF)-β signaling and is also known as a frequently mutated tumor suppressor gene in human pancreatic and colon cancer. However, considering the fact that TGF-β can contribute to cancer progression through transcriptional target genes, such as Snail, MMPs, and epithelial–mesenchymal transition (EMT)-related genes, loss of Smad4 in human cancer would be required for obtaining the TGF-β signaling-independent advantage, which should be essential for cancer cell survival. Here, we provide the evidences about novel role of Smad4, serum-deprivation-induced apoptosis. Elimination of serum can obviously increase the Smad4 expression and induces the cell death by p53-independent PUMA induction. Instead, Smad4-deficient cells show the resistance to serum starvation. Induced Smad4 suppresses the PAK1, which promotes the PUMA destabilization. We also found that Siah-1 and pVHL are involved in PAK1 destabilization and PUMA stabilization. In fact, Smad4-expressed cancer tissues not only show the elevated expression of PAK1, but also support our hypothesis that Smad4 induces PUMA-mediated cell death through PAK1 suppression. Our results strongly suggest that loss of Smad4 renders the resistance to serum-deprivation-induced cell death, which is the TGF-β-independent tumor suppressive role of Smad4.

Abstract 5234: Network interrogations on SNAIL inhibitor GN-25 induced perturbations in HMLE-SNAIL cell line models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC HMLEs (HMLE-SNAIL and Kras-HMLE-SNAIL pairs) serve as excellent model system to interrogate the effect of SNAIL targeted agents that reverse epithelial-to-mesenchymal transition (EMT). We developed a SNAIL-p53 interaction inhibitor (GN-25) that was shown to suppress SNAIL function. In this report, using pathway network analysis, we show that GN-25 induces the reversal of EMT to mesenchymal-to-epithelial transition (MET) in well recognized HMLE-SNAIL and Kras-HMLE-SNAIL models. GN-25 induced MET was found to be concurrent with growth inhibition, suppression of spheroid forming capacity and induction of apoptosis. We performed systems and pathway network analysis on mRNA expression by microarrays from GN-25 treated Kras-HMLE-SNAIL cells that showed an orchestrated global re-organization of EMT network genes. The expression signatures were validated at the protein level using confocal microscopy, western blot analysis and time lapse videography, and we found down-regulation of mesenchymal markers such as TWIST1, TWIST2, ZEB-1 concurrently with up-regulation of epithelial marker E-Cadherin. Additionally, RNAi studies validated SNAIL dependent mechanism of action of the drug. Most importantly, GN-25 deregulated many major transcription factors (TFs) such as inhibition of oncogenic TFs Myc, TBX2, NR3C1 and enhancement in tumor suppressor TFs such as SMAD7, DD1T3, CEBPA, HOXA5, TFEB, IRF1, IRF7 and XBP1, resulting in MET as well as induction of cell death. Our systems and network investigations provide solid pre-clinical supportive evidence for clinical application of GN-25 for the reversal of EMT phenotype to MET in aggressive cancers. Citation Format: Asfar S. Azmi, Aliccia Bollig-Fisher, Bin Bao, Bum Joon-Park, SH Lee, Gyu-Yong Song, Ramzi M. Mohammad, Fazlul H. Sarkar. Network interrogations on SNAIL inhibitor GN-25 induced perturbations in HMLE-SNAIL cell line models. [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 5234. doi:10.1158/1538-7445.AM2013-5234

Abstract P2-19-04: Latissimus dorsi muscle onlay patch alternative to acellular dermal matrix is useful for implant-based breast reconstruction

Introduction: The implants-based breast reconstruction is very useful method for breast conserving surgery in breast cancer. Recently, acellular dermal matrix (ADM) is applied to release the inferior pectoralis muscle for maintaining breast contour and to prevent burst out from the implant pocket. Authors applied latissimus dorsi (LD) muscle onlay patch instead of ADM for implant-based breast reconstruction. Patients and methods: From January to December 2012, a total of 20 patients with breast tumor underwent 22 cases of implant-based breast reconstruction with ADM or LD muscle onlay patch. The cosmetic outcome is assessed with 4-point scoring system at 4 weeks after from chemotherapy or radiotherapy. And the statistical analysis was evaluated between 2 groups. After conventional breast conserving surgery was done, the origin of the pectoralis major muscle are released and the window is checked pectoralis major muscle medially, rectus sheath inferiorly, and serratus anterior muscle laterally. Then, LD flap is sutured to cover this window with avoiding the vessel injury. The implant is inserted in the subpectoral pocket and breast reconstruction is finished with LD flap. Results: Mean operation time of ADM group and LD muscle onlay patch group was 153.9 and 299.7 minutes, respectively. (p = 0.054). There were no significant differences between 2 groups; postoperative complication, cosmetic outcomes, and tumor characteristics. However, ADM was found to be much more expensive than LD muscle onlay patch.(p Conclusion: Implant-based breast reconstruction with LD muscle onlay patch would be a feasible surgical technique alternative to ADM, when LD flap is planned for breast reconstruction. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-19-04.

Abstract B3: Induction of Smad4 in response to serum starvation promotes cell death through PUMA stabilization

Smad4 is an essential factor in TGF-β signaling and is also known as frequently mutated tumor suppressor gene in human pancreatic and colon cancer. However, considering the fact that TGF-β can contribute to cancer progression through transcriptional target genes such as Snail, MMPs, and EMT-related genes, loss of Smad4 in human cancer would be required for obtaining the TGF-β signaling independent advantage, which should be essential for cancer cell survival. Here we provide the evidence about novel role of Smad4, serum-deprivation-induced apoptosis. Elimination of serum can obviously increase the Smad4 expression and induces the cell death by p53-independent PUMA induction. Instead, Smad4 deficient cells show the resistance to serum starvation. Induced Smad4 suppresses the PAK1, which promotes the PUMA destabilization. We also found that siah-1 and pVHL are involved in PAK1 destabilization and PUMA stabilization. In fact, Smad4-expressed cancer tissues show the elevated expression of PAK1, also supprots our hypothesis that Smad4 induces PUMA-mediated cell death through PAK1 suppression. Our results strongly suggest that loss of Smad4 render the resistance to serum-deprivation-induced cell death, which is the TGF-β independent tumor suppressive role of Smad4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B3.

Abstract A50: Loss of pVHL inactivates p53 by permission of interaction between p14/ARF and progerin

Renal cell carcinoma (RCC) is a common primary renal malignancy in adults. The typical feature is the nuclear irregularity which is a basis for grading. Moreover, resistance to chemotherapy and irradiation (IR) is also one of the characters of RCC. p53 inactivation without genetic mutation and pVHL mutation in early stage are shown in RCC. However, it is still remained the question about the mechanism of the nuclear deformation and chemotherapy resistance. Here we proposed that the effect of progerin on RCC. Progerin elevated in RCC and the elimination of progerin could ameliorate the nuclear morphology. Moreover, knockdown of progerin could recover DNA damage sensitivity and p53 activation in RCC. Loss of pVHL induces elevated progerin expression, which inhibit p14/ARF by direct interaction to suppress p53 activity. Moreover, forced enhancement of progerin is found in human leukemia sample. In this study, we provide that the novel mechanism for p53 suppression in RCC and the importance of progerin in cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A50.

Immediate breast reconstruction with contralateral pectoralis major myomammary flap for breast conserving surgery.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5157 Purpose : The breast reconstruction techniques are mandatory in case of the indication permits for there has been lots of data showing no less survival as far as surgery keeps the principle of cancer surgery. It could be selected by many factors but mainly by breast size, site of tumor. The latissimus dorsi musculocutaneous flap (LDMCF) is one of the most commonly used techniques in early breast cancer patient who has small breast. But, it has difficulties to supply enough tissues to the widely excised tumor site. And especially in case of ptosis patient, reduction mammoplasty only is not enough to get the symmetry of the breast. We suggest a pectoralis major myomammary flap(PMMF) as a useful technique for the patients with ptosis. Methods : Seventeen patients with ptosis were treated with breast conserving using PMMF surgery. A quadrantectomy rather than lumpectomy was performed through a planned skin incision, and axillary lymph node dissection was performed according to the sentinel lymph node result. PMMF is harvested carefully without perforating branch injury of internal thoracic artery. And reconstruction was done by the PMMF through the medial tunnel between both breasts. Results : Among those of seventeen patients, seroma occurred in two patients and no necrosis was occurred. The cosmetic result showed good in fifteen and excellent in two patients based on four-point scoring system of breast cosmetics. Conclusions : After enough quadrantectomy to keep the cancer surgery principles, PMMF was quite useful to supply proper tissues for breast reconstructions, especially for the ptosis patient. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5157.