Molecular characterization of the zebrafish ff1b gene

Ph.DDOCTOR OF PHILOSOPH

Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not

Prostate cancer urine signatures.

<p>This composite data display illustrates the difference between Gleason scores (counts of B2M are included).</p

Urine RNA signatures of non-cancer.

<p>The histogram data display shows the signal counts for P08-022N (blue) and P08-027N (magenta). The high counts of B2M were excluded for data display. P08-022N shows no signals for the cancer markers. In contrast, P08-027N shows counts for these markers.</p

Correspondence between nCounter and DNA microarray analysis.

<p>Comparison of the raw signal values obtained for (a) PC3 and (b) C4-2/C4-2B cells are shown. The bottom panel (c) shows the comparison of fold-change calculated from the obtained data values.</p

Urine RNA signature of Gleason 4+5. A.

<p>The data display shows the high signal counts obtained, especially that of CD24. <b>B.</b> CD24 immunohistochemistry shows strongly stained cancer cells in tumor specimens 01-009E and 02-034A. Magnification is 40x.</p

Urine RNA signatures of cancer. A.

<p>The signal counts for P08-024pre (Gleason 3+3), P08-029pre (Gleason 3+3, T2c), P08-025pre (Gleason 3+4), and P08-046pre (Gleason 3+4, T2c) are compared to those of P08-022N. Note not only the cancer markers but also the prostate luminal markers produced signals (cancer cells also produce these markers). <b>B.</b> Two serial sections of tumor specimen 08-052A were stained for CCL3 and AGR2 expression. Magnification is 100x.</p

RNA amplification. A.

<p>Shown is Excel format of nCounter data from unamplified <i>vs.</i> amplified (marked by *) RNA of C4-2B and PC3 cells. POS and NEG are control probesets. <b>B.</b> Signal counts obtained from unamplified <i>vs</i>. amplified (marked by *) are compared in histogram display. Gene count for B2M is set at 100 and all other gene counts are compared to that of B2M from the same sample. Note genes with absent expression were not amplified.</p

Digital gene counts.

<p>The table shows the normalized gene counts in a dilution series of 10,000, 1,000, 100, 10, 1 (3x) cells of PC3 <i>vs</i>. C4-2. The genes are listed in the first column. The dataset query display for expression levels of these genes in C4-2 and PC3 is shown on the right.</p