Chromosomal and centrosomal Sgo1 isoforms have varying expression levels in different normal and cancerous tissues

2.1.3. Chromosomal and centrosomal Sgo1 isoforms have varying expression levels in different normal and cancerous tissues

Since overexpression as well as depletion of Sgo1 causes defects in both the chromosome and the centrosome cycles, the expression of the different isoforms of Sgo1 must be tightly regulated, since both, chromosomal and centrosomal aberrations can be tied to the development of cancer (Basto et al., 2008; Lingle et al., 2002; Pihan et al., 2001 and reviewed in Chan, 2011; Hanahan and Weinberg, 2011;

Santaguida and Amon, 2015). To test a possible correlation between the expression levels of the various Sgo1 variants and tumorigenesis, I performed comparative real time quantitative PCR (qPCR) using the TaqMan gene expressing assay with primers specifically recognizing either Sgo1 A1 (and C1) or A2 and C2, i.e. chromosomal or centromeric Sgo1, respectively.

figure 12. Expression of Sgo1 variants in different tissues.

(A) The PCR was conducted after a standard protocol for TaqMan gene expression assay, modified for the Sgo1 isoform specific primers.

(B) The amplification plot shows the variation of log (ΔRn) with PCR cycle number for the qPCR with Sgo1 A1 and A2/C2 specific primers on cDNA from testis, brain and fetal kidneys. The relative

RESULTS

!

figure 12 continued: normalized reporter value (ΔRn) is defined as the measured reporter value, normalized to the signal of an internal passive reference dye (ROX), minus the Rn value of the baseline signal generated by the instrument. To compare the actual expression levels, one has to set a threshold (in this case 0.202), which is a specific value of signal intensity at which all curves begin to indicate exponential signal increase.

(C) Relative expression of Sgo1 A1 and A2/C2 in the different tissues. The relative expression is defined as 2^-ΔCt. The cycle number, at which the signal reaches the threshold is called Ct value (for threshold cycle), from which the Ct value of the housekeeping gene (GAPDH) is subtracted, giving the

Δ^Ct value.

The expression level of GAPDH served as an internal standard. In preliminary experiments I established the optimal temperatures and number of cycles for the PCR reaction (figure 12A). Using human cDNAs from testis, brain and fetal kidney as templates, the fluorescence signals produced by the PCR reactions increased exponentially until they reached a plateau, which is caused by the limiting amount of primers (figure 12B). The relative expression of Sgo1 A1 and A2/C2 varied in the different tissues with the highest expression in the testis and the lowest expression in the brain (figure 12C). Note that doubling the amount of template resulted in about twice the relative expression, which verifies the fidelity of the assay. Interestingly, only in testis and fetal kidney the level of Sgo1 A1 exceeded that of Sgo1 A2 and C2.

In the brain however, Sgo1 A2/C2 level was about 4 times higher than the level of Sgo1 A1. Thus, expression levels of Sgo1 isoforms seem to be tissue specific.

To investigate the expression levels of Sgo1 in different cancer tissue samples, I used a commercially available cancer tissue array containing 96 samples of cDNA, which were obtained from 8 different tissues (breast, colon, kidney, liver, lung, ovaries, prostate and thyroid gland) at different stages of cancer. In addition three non-cancerous samples were included per tissue (figure 13, stage 0). In this array, the amounts of the pre-plated cDNAs were normalized to and validated with ß-actin, which means that all relative expression levels can be directly compared. While non-cancer samples (stage 0, figure 13) exhibited relative expression levels of both isoforms between 0.5 and 2, some samples especially from lung and ovary cancers reached expression levels up to 20 times higher (figure 13E-F). These numbers are only slightly lower for colon and breast cancer samples, which reach relative expression levels of up to 16 (figure 13G-H). While the correlation is much milder, also prostate, thyroid gland, kidney and liver tissues exhibited elevated Sgo1

!

figure 13. Expression of Sgo1 variants in different cancer tissue samples.

A1

stage 0 0 I I IIA IIA IIIAIIIAIIIC IV IV

mean rel. expression

0 0 0 I IIA IIA IIA III IIIBIIICIIIC IV

mean rel. expression

0 0 0 IA IB IC IIB IIIAIIIBIIICIIIC IV

mean rel. expression

RESULTS

!

figure 13 continued:

Measurement of the Sgo1 A1 and A2/C2 expression levels in cDNA from normal and cancer tissue samples of thyroid gland (A), prostate (B), kidney (C), liver (D), lung (E), ovaries (F), breast (G) and colon (H) using qPCR. The PCR reactions were performed as shown in figure 12A using “TissueScan”

cancer tissue cDNA arrays as templates. Shown are mean relative expression levels of Sgo1 A1 and A2/C2. Each set of tissue consists of 3 samples of normal tissues (stage 0) and 9 tissue samples from different stages of cancer (stage I-IV). Every column represents averages of three independent experiments. Bars represent standard deviation.

However, while there is a clear general link between all tested cancer types and Sgo1 overexpression, there is no apparent association between discrete levels of Sgo1 overexpression and specific stages of cancer. Many cancer samples exhibited simultaneous overexpression of centromeric (A1) as well as centrosomal Sgo1 (A2/C2), a notable example being the tissue sample from ovary cancer stage IC (figure 13F). Yet interestingly, in many samples centrosomal or centromeric Sgo1 isoforms seem to be differentially up-regulated. While, for example, many breast cancer-derived samples exhibited higher expression of centromeric compared to centrosomal Sgo1 (figure 13G), in several other samples, some of which are marked by dramatic Sgo1 overexpression (namely lung cancer stages IA and IIIA, and colon cancer stage I), the centrosomal Sgo1 isoforms heavily dominated over their centromeric counterpart. Distorted proportions were also observed for many of the cancer with weaker phentypes (especially in prostate tissues), which overexpressed mainly centrosomal Sgo1. Together these results not only argue that Sgo1 A2 and C2 most likely play a role in the development of cancer but also that tumorigenesis might be linked to the differential misregulation of centrosomal versus centromeric Sgo1 isoforms.