Earlier studies have documented that members of the Id family of proteins are expressed in solid tumors, yet their function and therapeutic value, especially in human CRC, remains largely unknown (Kleeff et al., 1998
; Rockman et al., 2001
; Wilson et al., 2001
; Lasorella et al., 2002
; Lofstedt et al., 2004
). The experiments described in this study demonstrate that Id2 represents a potential target in cancers where its expression occurs, and is critical for the growth of CRC cells. Id2 was not expressed in normal human colonic tissue. However, Id2 expression was present in greater than 40% of primary colon tumors and in almost all liver metastases studied, suggesting that Id2 may be a contributing factor for CRC metastasis. This observation is of particular clinical interest as the most common site of metastasis is the liver, and hepatic tumor burden is the most common cause of death in patients with metastatic CRC. These results, contrary to Id2
germline analysis where Id2
suppressed tumor formation (Russell et al., 2004
), suggesting that Id2 may have opposing functions in developmental and oncogenic tissues. Similar functions have also been described for other Rb regulatory proteins including E2F (Weinberg, 1996
). This dichotomy may be dependent upon the availability of additional co-regulators that direct Id2 function. Id2 was also expressed in all six of the human CRC cell lines assayed, and reduction of Id2 levels in CRC cells by siRNA decreased proliferation rates and increased spontaneous apoptosis. Further, our in vivo
models showed that knockdown of Id2 expression, through either the creation of stable Id2-deficient clones or the systemic administration of siRNA to Id2, severely impaired the ability of CRC cells to grow in the liver, the major site of CRC metastasis.
Consistent with findings in other oncogenic tissues, reduction of Id2 levels in CRC cells with siRNA decreased proliferation rates (Lasorella et al., 1996
). Earlier reports indicated that Id2 interferes with E2 transcription (Pagliuca et al., 2000
; Rothschild et al., 2006
) and Rb sequestration of E2F (Iavarone et al., 1994
; Lasorella et al., 2000
), both critical cell cycle regulators. Our examination of cell regulatory proteins revealed that reduction of Id2 levels in CRC cells downregulated cyclin D1 levels. These findings are contrary to those of an earlier study in which ectopic overexpression of Id2 in an Rb mutant osteosarcoma cell line decreased cyclin D1 levels (Lasorella et al., 1996
). In cells with functional Rb, such as HCT-116 cells (Broude et al., 2007
), cyclin D1 is required for cell cycle progression (Lukas et al., 1995
). The ability of Id2 expression to modulate cyclin D1 may depend on the presence of functional Rb and its coordination of cell cycle progression. We also showed that knockdown of Id2 increased levels of the cell cycle inhibitor p21 in the absence of changes in p53 levels (Lasorella et al., 1996
). Expression of p21 can be upregulated by members of the E2 family (Lasorella et al., 1996
; Prabhu et al., 1997
), which are antagonized by Id heterodimerization. Thus, decreasing Id2 levels could enhance E2 homodimerization, leading to upregulation of p21
in CRC cells. To further examine the relationship between Id2 and E2 gene regulation, we assessed levels of the E2 target gene Axin2
(Hughes and Brady, 2005
). Levels of Axin2 remained unchanged in HCT-116 cells regardless of Id2 levels (data not shown). This may suggest that other members of the Id family, whose levels remain unchanged in our Id2-siRNA-targeted cells, can regulate other aspects of E2 gene control.
In addition to its effect on cellular proliferation, we also showed that reduction of endogenous Id2 levels increased spontaneous apoptosis in CRC cells. This increase seems to be independent of p53, which remained unchanged in our Id2-deficient CRC cells (data not shown). Similar findings attributing anti-apoptotic functions to Id2 have been reported for T cells (Cannarile et al., 2006
) and prostate cancer, in which this effect was also independent of p53 (Asirvatham et al., 2007
). Examination of apoptotic regulatory proteins showed that reduction of Id2 resulted in increased levels of Bim/Bod, which antagonizes the survival functions promoted by Bcl-2 and its homolog Bcl-xl (Hsu et al., 1998
; O’Connor et al., 1998
). Our data suggests that Id2 represses Bim/Bod expression. The ability of Id2 to downregulate Bim/Bod levels may have a function in promoting colon cancer cell survival (Hsu et al., 1998
). Additionally, in our study the knockdown of Id2 expression increased cleavage of caspase-7. Although activation of this apoptotic pathway has not previously been linked to Id2 expression, loss of Id1 expression has been shown to increase poly (ADP-ribose) polymerase cleavage (Zhang et al., 2007
), itself a marker of apoptosis (Oliver et al., 1998
), suggesting that the Id proteins may function in promoting cell survival.
We also showed the importance of Id2 in CRC growth through the use of stable transfectants with reduced Id2 expression and by targeting Id2 in vivo
using liposomal-conjugated siRNA. Tumors arising from the stable cell lines lacking Id2 demonstrated reduced growth and tumorgenicity in both subcutaneous and in orthotopic (liver metastases) models. Tumors that did grow at either site were smaller than tumors derived from cells with normal Id2 levels. Similar results were achieved by targeting human CRC cells implanted in the livers of mice in vivo
with liposome-conjugated siRNA to Id2. Although the siRNA–DOPC complex is not specific for the human cancer cells implanted in the murine liver, we sought to determine if siRNA to Id2 had any effect on the mouse homolog of Id2. A Basic Local Alignment Search Tool (BLAST) homology search was performed and found that the regions targeted by the human siRNA utilized in our studies demonstrated no homology with the concomitant murine sequence; in addition, transient transfection with the human specific siRNAs did not reduce endogenous Id2 expression in a murine hepatic endothelial cell line (Langley et al., 2003
; data not shown). These findings suggest that Id2 liposomal siRNA designed to inhibit human Id2 in vivo
had no effect on murine Id2; thus we believe that the observed changes in tumor growth were mediated exclusively by blocking the Id2 expressed by the tumor cells.
In summary, our study here demonstrates that Id2 is an important mediator in CRC growth and metastasis, and a promising therapeutic target in tumors where its expression occurs. Our results indicate that Id2 enhanced cell proliferation, survival and in vivo tumor growth, all critical aspects of CRC progression. The use of anti-Id2 therapeutic targeting may improve existing targeted treatments and provides a unique opportunity to circumvent the ability of Id2-expressing tumors, such as CRC, to grow and metastasize.