AKT-mediated phosphorylation of Sox9 induces Sox10 transcription in a murine model of HER2-positive breast cancer.

BACKGROUND: Approximately 5-10% of HER2-positive breast cancers can be defined by low expression of the Ste20-like kinase, SLK, and high expression of SOX10. Our lab has observed that genetic deletion of SLK results in the induction of Sox10 and significantly accelerates tumor initiation in a HER2-induced mammary tumor model. However, the mechanism responsible for the induction of SOX10 gene expression in this context remains unknown. METHODS: Using tumor-derived cell lines from MMTV-Neu mice lacking SLK and biochemical approaches, we have characterized the signaling mechanisms and relevant DNA elements driving Sox10 expression. RESULTS: Biochemical and genetic analyses of the SOX10 regulatory region in SLK-deficient mammary tumor cells show that Sox10 expression is dependent on a novel -7kb enhancer that harbors three SoxE binding sites. ChIP analyses demonstrate that Sox9 is bound to those elements in vivo. Our data show that AKT can directly phosphorylate Sox9 in vitro at serine 181 and that AKT inhibition blocks Sox9 phosphorylation and Sox10 expression in SLK(-/-) tumor cells. AKT-mediated Sox9 phosphorylation increases its transcriptional activity on the Sox10 -7kb enhancer without altering its DNA-binding activity. Interestingly, analysis of murine and human mammary tumors reveals a direct correlation between the levels of active phospho-Sox9 S181 and Sox10 expression. CONCLUSIONS: Our results have identified a novel Sox10 enhancer and validated Sox9 as a direct target for AKT. As Sox10 is a biomarker for triple-negative breast cancers (TNBC), these findings might have major implications in the targeting and treatment of those cancers.

Periostin gene expression in neu-positive breast cancer cells is regulated by a FGFR signaling cross talk with TGFß/PI3K/AKT pathways.

BACKGROUND: Breast cancer is a highly heterogeneous disease with multiple drivers and complex regulatory networks. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. Postn has been shown to be involved in various processes of tumor development, such as angiogenesis, invasion, cell survival and metastasis. The expression of Postn in breast cancer cells has been correlated with a more aggressive phenotype. Despite extensive research, it remains unclear how epithelial cancer cells regulate Postn expression. METHODS: Using murine tumor models and human TMAs, we have assessed the proportion of tumor samples that have acquired Postn expression in tumor cells. Using biochemical approaches and tumor cell lines derived from Neu+ murine primary tumors, we have identified major regulators of Postn gene expression in breast cancer cell lines. RESULTS: Here, we show that, while the stromal compartment typically always expresses Postn, about 50% of breast tumors acquire Postn expression in the epithelial tumor cells. Furthermore, using an in vitro model, we show a cross-regulation between FGFR, TGFß and PI3K/AKT pathways to regulate Postn expression. In HER2-positive murine breast cancer cells, we found that basic FGF can repress Postn expression through a PKC-dependent pathway, while TGFß can induce Postn expression in a SMAD-independent manner. Postn induction following the removal of the FGF-suppressive signal is dependent on PI3K/AKT signaling. CONCLUSION: Overall, these results reveal a novel regulatory mechanism and shed light on how breast tumor cells acquire Postn expression. This complex regulation is likely to be cell type and cancer specific as well as have important therapeutic implications.

Recruitment and activation of SLK at the leading edge of migrating cells requires Src family kinase activity and the LIM-only protein 4.

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover and is regulated by the LIM domain-binding proteins Ldb1 and Ldb2. These adapter proteins have been demonstrated to interact with LMO4 in the organization of transcriptional complexes. Therefore, we have assessed the ability of LMO4 to also interact and regulate SLK activity. Our data show that LMO4 can directly bind to SLK and activate its kinase activity in vitro and in vivo. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding and Cre-mediated deletion of LMO4 in conditional LMO4(fl/fl) fibroblasts inhibits cell migration and SLK activation. Deletion of LMO4 impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Supporting this, Src/Yes/Fyn-deficient cells (SYF) expressing very low levels of LMO4 do not recruit SLK to the leading edge. Re-expression of wildtype Myc-LMO4 in SYF cells, but not a mutant version, restores SLK localization and kinase activity. Overall, our data suggest that activation of SLK by haptotactic signals requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. Furthermore, this establishes a novel cytosolic role for the transcriptional co-activator LMO4.

Expression of a kinase inactive SLK is embryonic lethal and impairs cell migration in fibroblasts.

Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLKK63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLKK63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLKK63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLKK63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development.

CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

SOX10 is a key regulator of melanoma progression and promotes a melanocytic/differentiated state. Here we identified melanoma cell lines lacking SOX10 expression which retain their in vivo growth capabilities. More importantly, we find that SOX10 can regulate T-cell infiltration in melanoma while also decreasing common cancer stem cell (CSC) properties. We show that SOX10 regulates CEACAM1, a surface protein with immunomodulatory properties. SOX10 directly binds to a distal CEACAM1 promoter region approximately 3-4kbps from the CEACAM1 transcriptional start site. Furthermore, we show that a SOX10-CEACAM1 axis can suppress CD8+ T-cell infiltration as well as reduce CSC pool within tumors, leading to reduced tumor growth. Overall, these results identify SOX10 as a direct regulator of CEACAM1, and uncover both a pro- and anti-tumorigenic roles for SOX10 in melanoma.

Muscle-specific deletion of SLK/Stk2 enhances p38 activity and myogenesis in mdx mice.

Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder characterized by the loss of dystrophin expression, muscle necrosis, inflammation and fibrosis. Ongoing muscle regeneration is impaired by persistent cytokine stress, further decreasing muscle function. Patients with DMD rarely survive beyond their early 20s, with cardiac and respiratory dysfunction being the primary cause of death. Despite an increase in our understanding of disease progression as well as promising preclinical animal models for therapeutic intervention, treatment options for muscular dystrophy remain limited and novel therapeutic targets are required. Many reports suggest that the TGFß signalling pathway is activated in dystrophic muscle and contributes to the pathology of DMD in part by impairing the differentiation of myoblasts into mature myofibers. Here, we show that in vitro knockdown of the Ste20-like kinase, SLK, can partially restore myoblast differentiation downstream of TGFß in a Smad2/3 independent manner. In an mdx model, we demonstrate that SLK is expressed at high levels in regenerating myofibers. Muscle-specific deletion of SLK reduced leukocyte infiltration, increased myogenin and utrophin expression and enhanced differentiation. This was accompanied by resistance to eccentric contraction-induced injury in slow fiber type-enriched soleus muscles. Finally, we found that these effects were partially dependent on the upregulation of p38 signalling. Collectively, these results demonstrate that SLK downregulation can restore some aspects of disease progression in DMD.

Loss of the Ste20-like kinase induces a basal/stem-like phenotype in HER2-positive breast cancers.

HER2 is overexpressed in 20-30% of all breast cancers and is associated with an invasive disease and poor clinical outcome. The Ste20-like kinase (SLK) is activated downstream of HER2/Neu and is required for efficient epithelial-to-mesenchymal transition, cell cycle progression, and migration in the mammary epithelium. Here we show that loss of SLK in a murine model of HER2/Neu-positive breast cancers significantly accelerates tumor onset and decreases overall survival. Transcriptional profiling of SLK knockout HER2/Neu-derived tumor cells revealed a strong induction in the triple-negative breast cancer marker, Sox10, accompanied by an increase in mammary stem/progenitor activity. Similarly, we demonstrate that SLK and Sox10 expression are inversely correlated in patient samples, with the loss of SLK and acquisition of Sox10 marking the triple-negative subtype. Furthermore, pharmacological inhibition of AKT reduces SLK-null tumor growth in vivo and is rescued by ectopic Sox10 expression, suggesting that Sox10 is a critical regulator of tumor growth downstream of SLK/AKT. These findings highlight a role for SLK in negatively regulating HER2-induced mammary tumorigenesis and provide mechanistic insight into the regulation of Sox10 expression in breast cancer.

Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness.

BACKGROUND: The Ste20-like kinase, SLK, plays an important role in cell proliferation and cytoskeletal remodeling. In fibroblasts, SLK has been shown to respond to FAK/Src signaling and regulate focal adhesion turnover through Paxillin phosphorylation. Full-length SLK has also been shown to be essential for embryonic development. In myoblasts, the overexpression of a dominant negative SLK is sufficient to block myoblast fusion. METHODS: In this study, we crossed the Myf5-Cre mouse model with our conditional SLK knockout model to delete SLK in skeletal muscle. A thorough analysis of skeletal muscle tissue was undertaken in order to identify defects in muscle development caused by the lack of SLK. Isometric force analysis was performed on adult knockout mice and compared to age-matched wild-type mice. Furthermore, cardiotoxin injections were performed followed by immunohistochemistry for myogenic markers to assess the efficiency muscle regeneration following SLK deletion. RESULTS: We show here that early deletion of SLK from the myogenic lineage does not markedly impair skeletal muscle development but delays the regenerative process. Interestingly, adult mice (~6 months) display an increase in the proportion of central nuclei and increased p38 activation. Furthermore, mice as young as 3 months old present with decreased force generation, suggesting that the loss of SLK impairs myofiber stability and function. Assessment of structural components revealed aberrant localization of focal adhesion proteins, such as FAK and paxillin. Our data show that the loss of SLK results in unstable myofibers resulting in a progressive myopathy. Additionally, the loss of SLK resulted in a delay in muscle regeneration following cardiotoxin injections. CONCLUSIONS: Our results show that SLK is dispensable for muscle development and regeneration but is required for myofiber stability and optimal force generation.