Subcellular localizations of IRS1, p-IRS1 and ß-catenin during Caco-2 polarization.

<p>Apotome immunofluorescence analysis at days 3, 7, and 14 postconfluency demonstrates differences in the cellular distribution of total IRS1 (IRS1, green), tyrosine 632-phosphorylated IRS1 (pIRS1, green), and ß-catenin (red) during the Caco-2 culture time course. For each field, the nuclei are counterstained in blue with 4′,6-diamidino-2-phenylindole (DAPI). Overlaps between red and green signals (merge) point to co-localizations (in yellow) of IRS1/pIRS1 and ß-catenin. Bar = 20 µm.</p

mRNA and protein levels of IRS1, <i>c-MYC</i>, insRß, IGF1R and ß-catenin in paired colonic mucosa and primary colorectal cancer (CRC).

<p>Panel A shows histograms of the relative expression of <i>IRS1</i> (left) and <i>c-MYC</i> (right) transcripts in paired samples of cancer-unaffected colorectal mucosa (white) and CRC (black), as determined by quantitative real-time PCR (RTqPCR). Mucosa samples were set equal to 100% and normalized to the relative expression of the housekeeping gene, <i>Cyclophilin</i>. Cancer samples were expressed relative to mucosa and normalized to the relative expression of the housekeeping gene. In pairs M1T1 to M4T4 and in M8T8, both <i>c-MYC</i> and <i>IRS1</i> increase in CRC relative to mucosa, only in M5T5 and M6T6 <i>IRS1</i> and <i>c-MYC</i> disagree (<i>IRS1</i>: <i>P</i> = 0.05, <i>c-MYC</i>: <i>P</i><0.001, unpaired t test on the means of all differences, data not shown). Panel B shows western blot analysis of IRS1, beta subunit of the insulin receptor (InsRß), beta subunit of the insulin-like growth factor 1 receptor (IGF1Rß), ß-catenin and ß-actin, as loading control, in the paired colonic mucosa and CRC samples shown in A (except M6T6, for which tissue for western blot analysis was not available). The histograms in Panel C show quantitations, after normalization for ß-actin, of the IRS1, InsRß, IGF1Rß and ß-catenin signals. Relative to paired mucosa, IRS1 is overexpressed in the CRCs of pairs M1T1-M4T4, together with InsRß, IGF1Rß and ß-catenin (IRS1: <i>P</i> = 0.017, InsRß: <i>P</i> = 0.044, IGF1Rß: <i>P</i><0.001, ß-catenin: <i>P</i><0.001, unpaired t test on the means of all differences, data not shown). Notably, the CRCs that overexpressed the IRS1, InsRß, IGF1Rß and ß-catenin proteins also overexpressed <i>IRS1</i> and c-<i>MYC</i> mRNA.</p

Expression of IRS1, insulin receptor, IGF1 receptor and ultrastructural differentiation in polarizing Caco-2 cells.

<p>Panel A shows western blot analysis of IRS1, beta subunit of the insulin receptor (InsRß), beta subunit of the insulin-like growth factor 1 receptor (IGF1Rß) and ß-actin, as loading control, in Caco-2 cells at days 3, 7 and 14 post-confluence, duplicated in absence (−) and presence (+) of serum in the culture medium. The histograms show quantitations, after normalization for ß-actin, of the IRS1, InsRß and IGF1Rß signals (means ± SE from the two experiments). Under both culture conditions increased espression of IRS1 and InsRß is clearly evident in polarized cells at day 14 (IRS1) and at days 7 and 14 (InsRß), whereas maximum expression of IGF1Rß is detected only at day 3. Transmission electron microscopy of Caco-2 cells at day 3 of the spontaneous polarization time course reveals forming electron-dense junctions at the apex of the lateral membranes of adjacent cells (panel A, arrow). With progression of polarization, tight junctions and desmosomes (panels C–D, arrows) and adhesion junctions (panel D) become evident as electron-dense plaques on adjacent lateral membranes at days 7 and 14, respectively. In addition, tight multicellular clusters, with differentiation features, such as intracellular lumina rich of apical brush border (panels E–F), become evident at day 14. Abbreviations: tj, tight junction; ad, adhesion junction; ds, desmosome. Panel G shows western blot levels of tyrosine 632-phosphorylated IRS1 (IRS1tyr632) and, as loading control, ß-actin, in serum-starved Caco-2 cells unstimulated (−) and stimulated (+) with insulin (100 nM) or IGF1 (10 nM). IRS1 tyrosine phosphorylation is relevant at day 7 of polarization, independently from the addition of exogenous insulin or IGF1. However, at day 3, only exogenous IGF1 determines IRS1 phosphorylation.</p

Density of IRS1 immunostaining in paired colonic epithelium, primary colorectal cancer and synchronous liver metastasis.

<p>Density values (mean ± SEM), normalized per area size in square pixels, were obtained by digital analysis using ImageJ software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) for IRS1 staining in: bottom versus top colonic crypt epithelium (respectively 11 and 10 areas); total colonic epithelium (78 areas) versus primary CRC (84 areas); total colonic epithelium (78 areas) versus liver metastasis (84 areas); primary CRC versus liver metastasis (84 areas). Only the differences between total colonic epithelium and liver metastasis and between primary CRC and liver metastasis are significant (<i>P</i><0.01).</p>*<p>Number of examined areas, CRC: colorectal cancer.</p

IRS1 and tumor histotype in primary colorectal cancer.

<p>Panels A and B respectively show diffuse cytoplasmic IRS1 in non-mucinous colorectal CRCs, including a moderately differentiated tumor, with strong immunostaining of cancer cells, and a poorly differentiated tumor, with weaker and possibly also nuclear IRS1 (arrowheads). Panels C–E show a poorly differentiated CRC with mucinous, mostly signet-ring phenotype. Notably, in the marginal area (single asterisk) detailed in panel D, tumor cells with non-mucinous phenotype show nuclear/perinuclear IRS1 (arrowheads), whereas signet-ring cells floating in mucin (double asterisk), detailed in panel E, do not show IRS1 immunostaining.</p

Spearman's correlations among IRS1, Ki67, p53, EGFR and ß-catenin in the primary colorectal cancer cases (n = 163).

<p>Abbreviations: ßCat: ß-catenin; EGFR: epidermal growth factor receptor; M: membrane staining; C: cytoplasmic staining; N: nuclear staining; Rho: Spearman's coefficient correlation. Significant correlations (<i>P</i><0.05) in bold.</p

Expression levels of IRS1, insulin receptor, IGF1 receptor, ß-catenin and ultrastructural differentiation in polarizing HT29 cells.

<p>Panel A shows western blot analysis of IRS1, beta subunit of the insulin receptor (InsRß), ß-catenin, beta subunit of the insulin-like growth factor 1 receptor (IGF1Rß), and ß-actin, as loading control, in HT29 cells maintained in complete medium during spontaneous differentiation at days 3 (pre-confluent), 7 (confluent) and 14 (post-confluent). The histograms show quantitations, after normalization for ß-actin, of the IRS1, InsRß, ß-catenin and IGF1Rß protein signals (means ± SE from two independent experiments). Expression of IRS1 and IGF1Rß is highest at day 3 and markedly declines at days 7 and 14, whereas InsRß is maximally expressed at day 14. At day 3, transmission electron microscopy of HT29 cells reveals bundles of intermediated filaments converging towards the plasma membrane to form electron-dense junctions between adjacent cell membranes (panel B–B1, arrow). With progression of the time-course, HT29 cells display differentiated features, such as desmosomes at days 7 and 14 (panel C–C1, arrow, and D–D1, arrow) and tight junctions at day 14 (D–D1, arrow). Abbreviations: tj, tight junction; ad, adhesion junction; ds, desmosome.</p

IRS1 immunostaining in cancer-uninvolved colonic epithelium, primary colorectal cancer and paired synchronous liver metastases.

<p>Panel A shows IRS1 immunostaining in full-length longitudinal sections of cancer-uninvolved colonic crypts. Panels B–E provide an example of IRS1 immunostaining in primary CRC (B–C) versus paired metastasis (liver biopsy core, D–E). Both show diffuse cytoplasmic IRS1, with much stronger immunostaining in metastatic cells. Panel F shows the histograms of the mean percentages of IRS1-positive cells in 24 cases of matching non-neoplastic colon epithelium, primary CRC and metastatic CRC (error bars mean ± SEM). There were significant differences between colonic epithelium (59.1±5.6%) and primary CRC (80.8±6.2%, <i>P</i> = 0.013 by independent sample t test) and between epithelium (59.1±5.6%) and hepatic metastasis (81.3±6.6%, <i>P</i> = 0.014). The difference between primary and metastatic CRC was not significant (<i>P</i> = 0.964).</p