טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPrinz Elad
SubjectThe Physiological Role of the JNK Scaffold Protein WDR62
in Cellular Signaling and Cancer
DepartmentDepartment of Medicine
Supervisor PROF. Ami Aronheim
Full Thesis textFull thesis text - English Version


Abstract

The mitogen-activated protein kinases (MAPKs) regulate a variety of cellular processes. A typical MAPK cascade is composed of MAP3K-MAP2K-MAPK kinases. The three main MAPK cascades are the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases. The JNK pathway is activated by various stresses, and is involved in regulation of diverse cellular processes, including proliferation, differentiation, survival and apoptosis. JNK plays an important role in human diseases such as cancer, diabetes, autoimmune and neurodegenerative diseases. A group of proteins that contribute to JNK specificity and signal efficacy are the scaffold proteins, which assemble the protein tier. WD40 repeats 62 (WDR62) is a JNK scaffold protein, interacting with JNK, MKK7 and several MAP3Ks. The loss of WDR62 in human leads to microcephaly and pachygyria. Yet, the role of WDR62 in cellular function is not fully studied. We hypothesized that WDR62 is required to coordinate JNK activity in response to specific stimuli.

In addition to its proposed role as a JNK scaffold and microcephaly associated protein, there is evolving evidence that WDR62 plays a positive role in cancer progression. Cancer is the leading cause of death in Israel. WDR62 have been shown to participate in the progression of ovarian, gastric and lung cancers. However, the role of WDR62 in breast cancer growth and metastasis have not been studied yet. The second part of my thesis aimed to study the role of WDR62 in breast cancer progression.

Towards this end, we used the CRISPR/Cas9 and ShRNA approaches to establish a human breast cancer cell line MDA-MB-231 with WDR62 loss of function or knockdown, respectively, and studied the consequence to JNK signaling. We found that WDR62 specifically mediates tumor necrosis α (TNFα)-dependent JNK activation through the association with both the adaptor protein, TNF receptor-associated factor 2 (TRAF2), and the MAP3K protein, mixed lineage kinase 3 (MLK3). TNFα-dependent JNK activation is mediated by WDR62 in HCT116 and HeLa cell lines as well. MDA-MB-231 harboring WDR62-knockout (WDR62-KO) cells display increased resistance to TNFα-induced cell death. Collectively, WDR62 coordinates the TNFα receptor signaling pathway to JNK activation through association with multiple kinases and the adaptor protein TRAF2. 

In the second part of my study, we used an orthotopic xenograft model of breast cancer using the above WDR62-deficient MDA-MB-231 cells. We found that WDR62 has no effect on primary tumor growth. However, WDR62-deficiency have markedly reduced the capacity of tumors to spread to the lungs. To study the molecular consequences on metastasis, we found that WDR62 had no effect on blood and lymph vessel formation. In addition, WDR62 loss did not alter the ability of cells to directly seed the lungs. Using invasion migration assay in vitro, we found that the WDR62-knockdown (WDR62-KD) cells had reduced migratory activity compared with WT cells. We also found that Kallikrein-6 (KLK6), a serine protease with anti-tumor effect in breast cancer, is upregulated in WDR62-KD cells.

Collectively, my study reveals novel roles for the JNK scaffold protein WDR62 in JNK signaling and breast cancer. WDR62 mediates TNFα-dependent JNK activation and cell migration and metastasis of breast cancer cells.