ANKHD1 Silencing Induces S-Phase Arrest and Modulates Cell Cycle Gene Expression in Human Myeloma Cells

Abstract 1828

ANKHD1 is a multiple ankyrin repeats containing protein with a single KH domain. It is a large protein (∼ 280 kDa) derived from an 8 kb transcript. The ANKHD1 gene, present in human chromosome 5q31.3 as a single copy is ubiquitously expressed in normal human tissues and reported to be highly expressed in cancers, such as acute leukemia. Previous study showed higher expression of ANKHD1 in bone marrow plasma cells (CD138+) from Multiple Myeloma patients as compared to control (1) and it is also over expressed in multiple myeloma cell lines such as MM1S, MM1R, U266 and RPMI 8266 at both mRNA and protein level (2). However, the functional role of ANKHD1 in myeloma cells is unknown. In the present study, by silencing ANKHD1 gene expression in glucocorticoid resistant (U266) and sensitive (MM1S) myeloma cell lines, we studied its effect on cell cycle, proliferation and apoptosis. For gene silencing, specific shRNA-expressing lentiviral vector targeting the ANKHD1 gene and as negative control, sequence specific to Lac z gene were used. Cell growth was measured using the MTT colorimetric assay, whereas for apoptosis and cell cycle analysis Flow cytometry was used. Western blot and RTPCR were used for studying gene expression and protein levels, respectively. The results showed that lentiviral vector containing coding sequences for shRNA significantly downregulated ANKHD1 gene expression in Multiple Myeloma cells at the mRNA and the protein levels (p<0.05). Furthermore, we found that the cell cycle was arrested at S phase and the cell proliferation was significantly inhibited in both cell lines studied (p<0.05). However, ANKHD1 suppression did not induce apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. To address the mechanism of the antiproliferative effect of ANKHD1 silencing, we examined the effect of ANKHD1 inhibition on cell cycle-related gene expression and proteins. ANKHD1 suppression caused downregulation of CDKN1B (p27), CCNB1 (cyclin B1), CDC25, CCNE1 (cyclin E1) and WEE 1 gene expression. There was no significant change in CCNA2 (Cyclin A2), CDC20 expression at mRNA levels. On the other hand, expression of CDKN1A (p21),which inhibits cyclin dependent kinases (CDKs) and plays role in preventing proliferation, was highly upregulated in both the cell lines. At protein levels, expression of Cdk2,Cdk4, p27 (CDKN1B) and E2F1 was decreased in both the cell lines with almost complete inhibition of expression in U266 cells. Taken together, the above results suggest that accumulation of cells in S phase (S phase arrest) can be due to inhibition of CDKs which binds with cyclins and are responsible for progression of cell cycle. Further, this inhibition of CDKs could be associated to increased induction of (CDKN1A) p21 in both cell lines.

In conclusion, the present study demonstrates that the suppression of ANKHD1 potently inhibits proliferation and promotes cell cycle arrest without affecting rate of apoptosis in both glucocorticoid resistant as well as sensitive multiple myeloma cells. Also, as ANKHD1 suppression prevents S to G2/M progression, ANKHD1 protein might have role in cell cycle control by modulating cell cycle gene expression in intra S phase check point. The mechanisms modulating expression of these genes are under investigation. Further studies with combination of drugs that induce apoptosis and suppression of ANKHD1 may be an effective strategy for treatment of cancers, and therefore needed to be explored.