BRK Phosphorylates SMAD4 for proteasomal degradation and inhibits tumor suppressor FRK to control SNAIL, SLUG and metastatic potential

Abstract

RUNNING TITLE: BRK Regulates FRK and EMT via SMAD4 Phosphorylation and degradation. AbstractThe tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Interestingly, our search for signaling pathways regulated by BRK, a non-receptor protein tyrosine kinase that is up-regulated in ~80% of invasive ductal breast tumors, led us to discover that BRK competitively binds and phosphorylates SMAD4, and regulates TGF-β/ SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F), phosphorylates SMAD4 resulting in its recognition by the ubiquitinproteasome system, which accelerates SMAD4 degradation. In agreement, we also observed an inverse protein expression pattern of BRK and SMAD4 in a panel of breast cancer cell lines and breast tumors. Activated BRK mediated degradation of SMAD4 causes the repression of tumor suppressor genes FRK that was associated with increased expression of mesenchymal markers and decreased cell adhesion ability. Thus, our data suggest that combination therapies targeting activated BRK signaling may have synergized the benefits in the treatment of SMAD4 repressed cancers. Therefore, our data propose that combination therapies which includes targeting activated BRK signaling may synergize the benefits in the treatment of SMAD4 deficient cancers. Results Components of the TGF-β/SMAD pathway are potential BRK targetsBRK is overexpressed in most breast cancer cell lines and tumors(21) , (5), and importantly, BRK is activated in the plasma membrane of human breast tumors(22). To substantiate the overexpression of BRK in most major cancer types, we interrogated the gene expression database, GENT (Gene Expression across Normal and Tumors;http://medicalgenome.kribb.re.kr/GENT/reference.php). We found that the expression of BRK mRNA was significantly higher (p≤ 0.05) in all five cancer types that we queried compared to their respective non-cancerous tissues (Figure 1A). Having confirmed that BRK overexpression is prevalent in cancers, we next sought to identify BRK targets.In this study, we focused on the constitutively-active form of BRK, BRK-Y447F (termed BRK-YF from here on). We have previously demonstrated that BRK-YF displayed higher kinase activity than BRK-WT when ectopically and stably expressed in HEK293 cells(5). To decipher the role of activated BRK in cellular signal transduction pathways, we expressed GFP-tagged, or SNAP-FLAG tagged BRK-WT and BRK-YF constructs in HEK293 cells and evaluated their global kinase activity by analyzing cell lysates by Western blotting. When we visualized phosphorylated proteins using the PY20 anti-phosphotyrosine antibody, we confirmed that BRK-YF showed higher kinase activity than BRK-WT (Figure 1B).

P53 Gene Expression and Nitric Oxide Levels after Artemisinin-Caffeine Treatment in Breast, Lungs and Liver of DMBA-Induced Tumorigenesis

“…p53 is one of the highly mutated genes in human cancers, the mutant p53's structures and functions are altered from being a proto-oncogene to an oncogene (Ghosh et al, 2022). Mutant p53 interacts with transcriptional factors or alters pathways such as SMAD/TGF-β signalling pathway, leading to uncontrolled cell proliferation and metastasis (Miah et al, 2019). Mutations, overexpression or non-functional alterations occur in p53 gene in over 50% cancers, and are associated with different cancers (Perri et al, 2016).…”

Transforming growth factor‐β signalling in tumour resistance to the anti‐PD‐(L)1 therapy: Updated

“…The TGFβ/SMAD network exerts complex biological activities. 21 TGFβ signalling is negatively regulated by SMAD7. In fact, SMAD7 restricts PD-1-induced regulatory T-cell (Treg) differentiation and limits responses from T cells to TGFβ, which further result in increased intestinal inflammation and progression of colitis.…”

“…62 The activity of TGFβ/SMAD is critical for promotion towards acquisition of EMT and metastasis. 21,63 TGFβ is released from TAMs, 30 and there is a positive cross-talk between TAMs with mesenchymallike tumour cells for promotion of metastasis of breast 64 and CRC cells. 65 Chronic exposure to the TGFβ promotes a stable EMT state in mammary carcinoma cells accompanied by stable generation of cancer stem cells (CSCs) and drug resistance that cannot be reversed after withdrawal of TGFβ, but it is responsive to the mTOR inhibition.…”

“…SMAD4 is the key mediator in the TGF‐β pathway; upon activation of TGF‐β, complexes of SMAD are formed through bondage of SMAD2 and SMAD3 with SMAD4. The TGF‐β/SMAD network exerts complex biological activities 21 . TGF‐β signalling is negatively regulated by SMAD7.…”

“…Zeb, 61 Snail and Slug (Snail2) are transcription factors related to the EMT profile in tumour cells 62 . The activity of TGF‐β/SMAD is critical for promotion towards acquisition of EMT and metastasis 21,63 . TGF‐β is released from TAMs, 30 and there is a positive cross‐talk between TAMs with mesenchymal‐like tumour cells for promotion of metastasis of breast 64 and CRC cells 65 .…”