Case Study 1: Developing a role statement for programme leadership

This case-study describes the collaborative development and evolution of Griffith University's Program Director's role statement and in particular the representation of the change nature of the role's leadership orientation as evident in its various iterations

Enrichment of FRA1 in tumor cells at the invasive front of human CRCs.

<p>(A) Low power image of a representative colorectal carcinoma stained with an antibody detecting FRA1. The asterisk indicates the lumen, while the arrowheads indicate the deep invasive front. Scale bar represents 1 mm. (B and C) High power images of the tumor centre (TC) and invasive front (IF) shown in (A). Arrowheads indicate tumor buds. Scale bar represents 10 µM. (D) Relationship between the intensity of nuclear FRA1 expression and the tumor budding marker, cytokeratin AE1/AE3 in 25 CRC cases.</p

FRA1 knockdown suppresses mesenchymal-like features in CRC cells.

<p>(A) Immunoblot analysis of FRA1 and E-cadherin levels in BE cells stably transduced with a non-silencing control (shNS) or FRA1-targeting shRNAs (shFRA1-A and -B). (B) Effect of FRA1 knockdown on basal and c-Jun-induced AP-1 reporter gene activity in BE cells. (C) Phase-contrast images (top row) and immunofluorescence staining for DAPI with ZO-1 (middle row) or vimentin (bottom row) on the cells from (A). Scale bar represents 10 µM. (D–F) Analysis of <i>in vitro</i> migration, invasion and proliferation in cells from (A). Error bars represent S.E.M. for 3 independent experiments.</p

Clinical significance of FRA1 and FRA1^EMT genes in CRC.

<p>(A) Kaplan-Meier plots of recurrence-free survival in stage B and C CRC patients according to expression of the FRA1 gene (<i>FOSL1</i>). (B) Unsupervised clustering of stage B and stage C CRCs based on FRA1^EMT genes encompassing concordant probesets exhibiting significant expression differences between the two main groups. Clustering divides cancers into groups with mesenchymal and epithelial profiles. Samples are arranged along the X-axis and genes along the Y-axis. Genes are grouped into those downregulated (blue) or upregulated (orange) upon FRA1 knockdown in BE cells relative to the mean- and sample-centered scaled expression. (C) Kaplan-Meier plots of recurrence-free survival in stage B and C CRC patients based on expression of both <i>FOSL1</i> (low vs high) and mesenchymal (Mes, dark green) or epithelial (Epi, light green) subsets of FRA1^EMT genes. The log-rank test was used for comparisons.</p

Characterization of EMT-related FRA1 transcriptional targets.

<p>(A) Heat map showing different functional groups of EMT-related genes bound and regulated by FRA1 (FRA1^EMT genes). Data from RNA-Seq analysis of two clones of BE shFRA1-A cells (n = 4 for each cell line) was normalised relative to shControl cells. Regions shown in red represent genes associated with an epithelial state that were upregulated upon FRA1 silencing (log fold-change&lt;−1, p&lt;0.05), while green regions represent mesenchymal-type genes repressed by FRA1 silencing (log fold-change&gt;1, p&lt;0.05). (B) Distribution of genomic FLAG-FRA1 binding sites identified by ChIP-Seq relative to a corresponding gene. The number of reads identified for each region is expressed as a percentage. (C) ChIP-qPCR analysis of FLAG-FRA1 binding to genomic regions in selected FRA1^EMT genes. Data represent relative enrichment compared to parental BE cells. A region of the miRNA-21 gene not bound by FLAG-FRA1 was used as negative control (<i>CTRL</i>). (D) qRT-PCR analysis of selected FRA1^EMT genes in BE cells stably transduced with one of two independent shRNAs targeting FRA1. Data are represented relative to expression levels in cells shNS cells. Student's t-test was used for all comparisons (^*p&lt;0.05, ^**p&lt;0.01, ^***p&lt;0.001). Error bars represent S.E.M. for 3 independent experiments. (E) FRA1 protein levels and (F) expression of epithelial and mesenchymal marker genes in a panel of CRC cell lines.</p

A FRA1-dependent autocrine TGFβ2 loop promotes mesenchymal gene expression in BE CRC cells.

<p>(A and B) Expression of selected mesenchymal (<i>TGFBI</i>, <i>AXL</i>) and epithelial (<i>CDH1</i>, <i>CLDN7</i>) FRA1^EMT genes upon transient knockdown of the TGFβ pathway FRA1 targets <i>TGFB2</i> and <i>SMAD3</i> using siRNA pools in BE cells. Data are represented relative to levels of these genes in cells transfected with siRNAs targeting GFP. (C) Effects of the TGFβ receptor inhibitor SB43152 (10 µM for 72 h) on expression of a selected mesenchymal FRA1^EMT (<i>TGFBI</i>, <i>AXL</i>) genes in BE cells. Student's t-test was used for all comparisons (^*p&lt;0.05, ^**p&lt;0.01, ^***p&lt;0.001). Error bars represent S.E.M. for 3 independent experiments.</p

FRA1 controls pro-mesenchymal transcriptional responses induced by TGFβ in CRC cells.

<p>(A) Immunoblot analysis of endogenous FRA1 expression in BE and SW837 CRC cells. (B) qRT-PCR analysis comparing relative expression levels of a selection of mesenchymal (<i>AXL</i>, <i>VIM</i>) and epithelial (<i>CDH1</i>, <i>CLDN7</i>) FRA1^EMT genes in BE and SW837 CRC cells. (C) Effects of transient FRA1 knockdown on TGFβ1-induced (10 ng/mL 48 h) expression of <i>VIM</i>, <i>CDH1</i> and <i>CLDN7</i> in SW837 cells. Student's t-test was used for all comparisons (^*p&lt;0.05). Error bars represent S.E.M. for 3 independent experiments.</p