Regulation of Epithelial Cell Plasticity by a SUMO-TGFβ Signaling Axis

Background Protein post-translational modification by the small ubiquitin-like modifier (SUMO), or SUMOylation, can regulate the stability, subcellular localization or interactome of a protein substrate with key consequences for cellular processes including the Epithelial-Mesenchymal Transition (EMT). The secreted factor Transforming Growth Factor beta (TGFβ) is a potent inducer of EMT in development and homeostasis. Importantly, the ability of TGFβ to induce EMT has been implicated in promoting cancer invasion and metastasis, resistance to chemo/radio therapy and maintenance of cancer stem cells. Interestingly, TGFβ-induced EMT and the SUMO system intersect with important implications for cancer formation and progression, and novel therapeutics identification. The transcriptional coregulator Ski-related novel protein N (SnoN), a negative regulator of TGFβ signaling axis, is a SUMO substrate. Interestingly, Protein Inhibitor of Activated STAT 1 (PIAS1) and Transcriptional Intermediary Factor 1 gamma (TIF1γ) are two distinct SUMO E3 ligases that bind and promote the SUMOylation of SnoN to suppress TGFβ-induced EMT. PIAS1 has been shown to act in a SUMO E3 ligase-dependent manner to suppress the invasion and metastatic growth of breast cancer cells. These results raised the key questions of the significance of the role of the two distinct SUMO E3 ligases PIAS1 and TIF1γ in regulating SnoN SUMOylation and suppressing TGFβ-induced EMT in mammary non-transformed epithelial cells and breast carcinomas. Hypothesis I hypothesize that PIAS1 and TIF1γ cooperate to promote SnoN SUMOylation to suppress EMT by the TGFβ-Smad pathway with potential relevance for breast cancer metastasis and prognosis. Results In this thesis, evidence is provided suggesting that the protein abundance and nuclear localization of PIAS1 act as survival biomarkers in breast cancer patients in a tissue microarray analysis. Accordingly, further results indicate that PIAS1 acts via SUMOylation of SnoN to suppress the invasive growth of triple negative breast cancer cells in 3D-organoid culture. In other studies, findings are provided that support the idea that SnoN promotes the formation of PIAS1-SnoN-TIF1γ multiprotein complex, which promotes SnoN SUMOylation, and its ability to suppress TGFβ-induced EMT in breast epithelial and cancer cells in 3D. Mechanistic studies suggest that SUMOylation promotes SnoN binding to the epigenetic regulators histone deacetylase 1 (HDAC1) and histone acetylase p300 in such a manner that leads to the suppression of EMT induced by the TGFβ-Smad-pathway in non-transformed and cancerous mammary cell-derived 3D organoids. Conclusions The novel findings in this thesis reveal the importance of a SUMO E3 ligase complex comprising PIAS1 and TIF1γ that enhances the SUMOylation of SnoN with impact on specific epigenetic regulators that control EMT in normal and cancer cells. These findings can lead to the discovery of novel biomarkers and therapeutics in breast and potentially other epithelial cell-derived cancers

The SUMO System and TGFβ Signaling Interplay in Regulation of Epithelial-Mesenchymal Transition: Implications for Cancer Progression

Protein post-translational modification by the small ubiquitin-like modifier (SUMO), or SUMOylation, can regulate the stability, subcellular localization or interactome of a protein substrate with key consequences for cellular processes including the Epithelial-Mesenchymal Transition (EMT). The secreted protein Transforming Growth Factor beta (TGFβ) is a potent inducer of EMT in development and homeostasis. Importantly, the ability of TGFβ to induce EMT has been implicated in promoting cancer invasion and metastasis, resistance to chemo/radio therapy, and maintenance of cancer stem cells. Interestingly, TGFβ-induced EMT and the SUMO system intersect with important implications for cancer formation and progression, and novel therapeutics identification

The transcriptional regulator SnoN promotes the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain

Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursor

Identification of the SUMO E3 ligase PIAS1 as a potential survival biomarker in breast cancer.

Metastasis is the ultimate cause of breast cancer related mortality. Epithelial-mesenchymal transition (EMT) is thought to play a crucial role in the metastatic potential of breast cancer. Growing evidence has implicated the SUMO E3 ligase PIAS1 in the regulation of EMT in mammary epithelial cells and breast cancer metastasis. However, the relevance of PIAS1 in human cancer and mechanisms by which PIAS1 might regulate breast cancer metastasis remain to be elucidated. Using tissue-microarray analysis (TMA), we report that the protein abundance and subcellular localization of PIAS1 correlate with disease specific overall survival of a cohort of breast cancer patients. In mechanistic studies, we find that PIAS1 acts via sumoylation of the transcriptional regulator SnoN to suppress invasive growth of MDA-MB-231 human breast cancer cell-derived organoids. Our studies thus identify the SUMO E3 ligase PIAS1 as a prognostic biomarker in breast cancer, and suggest a potential role for the PIAS1-SnoN sumoylation pathway in controlling breast cancer metastasis

Recombinant human PRG4 (rhPRG4) suppresses breast cancer cell invasion by inhibiting TGFβ-Hyaluronan-CD44 signalling pathway.

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFβ) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFβ-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFβ-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFβ has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFβ has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFβ actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer

Characterization of the PIAS1 antibody.

<p>(A) PIAS1 and actin-immunoblots of lysate of 293T cells expressing one of two different concentrations (1X or 10X) of FLAG-tagged PIAS1, PIAS2-xα, PIAS2-xβ, PIAS3 or, PIAS4, or transfected with the vector control. Actin immunoblotting was used as loading control. Only PIAS1 antibody-immunoreactive bands corresponding to endogenous PIAS1 and exogenous FLAG tagged-PIAS1 were detected. Immunoblots are from an experiment that was repeated twice with similar results. (B) PIAS1 or actin immunoblots of lysate of MDA-MB-231 cells transiently transfected with an RNAi vector control or receiving a pool of plasmids expressing short hairpin RNAs against two distinct regions of PIAS1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177639#pone.0177639.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177639#pone.0177639.ref019" target="_blank">19</a>]. Control and PIAS1 RNAi plasmids also express CMV-driven green fluorescence protein (GFP). Immunoblots are from an experiment that was repeated twice with similar results. (C) Representative PIAS1 (red), GFP (green) and nuclei (blue) fluorescence microscopy micrographs of MDA-MB-231 cells transfected as in B, and subjected to anti-PIAS1 indirect immunofluorescence and counterstained with Hoechst 33342 fluorescent nucleotide dye to visualize nuclei. GFP signal indicate control vector or PIAS1 RNAi plasmid-transfected cells. Arrow shows an example of each a vector transfected cell (upper row) and a PIAS1 RNAi transfected cell (lower row) to highlight the knockdown of endogenous PIAS1 in PIAS1i-transfected cell. These experiments were repeated two times with similar outcomes. (D) Representative PIAS1 immunoblots of serially-diluted lysate of 293T cells (upper panel), and protein abundance of PIAS1 quantified by densitometry (y-axis) plotted versus the protein amount of lysate (x-axis) (lower panel). The abundance of PIAS1 for each point in the XY graph is the mean ± SEM from three independent experiments including the one shown in upper panel. Regression analysis indicated that the protein abundance of PIAS1 follows a linear relationship with total protein amount in cells lysates.</p

Endogenous SnoN mediates the ability of PIAS1 to suppress the invasive growth of breast cancer cell-derived organoids.

<p>(A) SnoN and actin immunoblots of lysate of MDA-MB-231 cells cotransfected with a plasmid encoding a short hairpin RNA targeting a specific region of SnoN mRNA or a control plasmid, together with an expression plasmid encoding an RNAi-resistant SnoN (SnoN(r)) or wild type RNAi-sensitive SnoN, or the corresponding vector control. (B) Representative DIC light microscopy micrographs of untreated or 100pM TGFβ-treated 8-day old three-dimensional organoids derived from MDA-MB-231 cells stably expressing PIAS1 or the corresponding vector control, and co-transfected with SnoN RNAi, SnoN(r), wild type SnoN, or respective control plasmids. (C) Bar graph depicts mean ± SEM proportion of non-deformed organoids expressed as a percentage of total colonies counted for each experimental condition from three independent experiments including the one shown in B. Non-deformed organoids represents non-invasive growth phenotype. Knockdown of endogenous SnoN promoted invasive growth of organoids as compared to vector control or PIAS1 stably expressing cells in the absence or presence of TGFβ. Expression of SnoN(r) but not wild type SnoN suppressed the ability of SnoN RNAi to promote invasive growth of organoid in vector control or PIAS1 expressing cells in the absence or presence of TGFβ. Significant difference, ANOVA: ***P≤0.001. Scale bar indicates 50 μm. Arrows and arrowheads indicate non-deformed and invasive organoids, respectively.</p

Sumoylation of SnoN supresses TGFβ-induced invasiveness of breast cancer organoids.

<p>(A) SnoN and actin immunoblots of lysates of MDA-MB-231 cells expressing SnoN (WT), SnoN (KdR), SUMO-SnoN or transfected with a control vector. (B) Representative DIC light microscopy micrographs of untreated or 100pM TGFβ-treated 8-day old three dimensional organoids derived from MDA-MB-231 cells transfected as in A. (C) Bar graph represents mean ± SEM proportion of non-deformed organoids expressed as a percentage of total colonies counted for each experimental condition from four independent experiments including the one shown in B. Non-deformed organoids represents non-invasive growth phenotype. SnoN (KdR) promoted an invasive growth of breast cancer cell-derived organoids even in the absence of TGFβ. SUMO-SnoN suppressed TGFβ-induced invasive growth of breast cancer cell-derived organoids. Significant difference, ANOVA: ***p<0.001. Scale bar indicates 50 μm. Arrows and arrowheads indicate non-deformed and invasive organoids, respectively.</p

Correlation of clinicopathological characteristics and DSOS in Calgary 65/65 cohort.

<p>Correlation of clinicopathological characteristics and DSOS in Calgary 65/65 cohort.</p

PIAS1 protein abundance analysis in reference TMA and breast cancer TMA.

<p>(A) PIAS1 and actin immunoblots of lysate of MDA-MB-231 cells expressing four increasing concentrations of PIAS1 (samples 1 to 4). (B) Bar graph of actin-normalized PIAS1 protein abundance in samples 1 to 4 shown in A and expressed relative to actin-normalized PIAS1 abundance in sample 3. (C) PIAS1 immunoblot of serially diluted lysate of MDA-MB-231 cells overexpressing PIAS1 in sample 4. (D) XY-graph plot of the lysate's total protein on the x-axis versus PIAS1 protein abundance, quantified from PIAS1immunoblot in C, on the y-axis. (E) Representative PIAS1 (red), and nuclei (blue) fluorescence micrographs of sections of Histogel-embedded MDA-MB-231 cells reference TMA expressing increasing abundance of PIAS1, corresponding to samples 1 to 4 in panel A, which were subjected to anti-PIAS1 indirect immunofluorescence and DAPI dye staining to visualize nuclei. (F) Bar graph depicts AQUA analysis software-quantified PIAS1 abundance in the reference TMA shown in E. (G) The XY-graph shows the relationship between Log of relative abundance of PIAS1 in samples 1 to 4 of MDA-MB-231 cell lysates quantified by immunoblotting on the x-axis versus Log of abundance of PIAS1 in MDA-MB-231 samples 1 to 4 of reference TMA quantified by AQUA analyses of immunocytochemistry on the y-axis. (H) Representative fluorescence microscopy micrographs of histogel-MDA-MB-231 cell reference TMA blocks. (I) Representative fluorescence microscopy micrographs of paraffin-embedded normal breast tissue and examples of three breast cancer tissues expressing different amounts of PIAS1. For both H and I, TMA were subjected to anti-PIAS1 (red) and anti-Pan cytokeratin (green) antibodies indirect immunofluorescence, and nuclear counterstaining with DAPI (blue). PIAS1-Cytokeratin-Nuclei merged fluorescence micrograph panels show relative abundance of PIAS1 in reference breast cancer TMA, normal breast and breast cancer tissue array.</p