GeneLoc location for GC08M012985:
Start: 12940872 bp from pter,
End: 13372395 bp from pter,
Size: 475895 bases,
Orientation: minus strand.

DLC1 was first isolated from human hepatocellular carcinoma (HCC) by PCR-based subtractive hybridization method in 1998 (Yuan et al., 1998). It maps to human chromosome 8p21.3-22, a region recurrently deleted in HCC and other solid tumors. Several isoforms of DLC1 have been found, namely DLC1alpha (isoform 2), DLC1beta (isoform 1) and DLC1gamma (isoform 3). DLC1alpha, DLC1beta and DLC1gamma contain 15 exons, 19 exons and 6 exons respectively.

Transcript size:
DLC1alpha: 6044 bp (NM_006094),
DLC1beta: 7445 bp (NM_182643),
DLC1gamma: 1975 bp (NM_024767).
Among the three DLC1 isoforms, DLC1alpha represents the predominant isoform. It has a unique 5 untranslated region and a transcript size of 6044 bp. DLC1beta has the longest transcript size of 7445 bp. It contains a total of 19 exons including exons 2 to 14 of DLC1alpha and another 5 exons upstream of the transcriptional start site of DLC1alpha. DLC1gamma is the shortest isoform with a transcript size of 1975 bp. It shares the first 5 exons with DLC1gamma and has a unique exon 6.
CpG islands have been reported to be found in -387 to +502 and +554 to +765 of DLC1alpha promoter. In this region, 11 SP1 and 4 GCF transcription factor binding sites (-162 to +67) have also been documented (Yuan et al., 2003). Genomic deletions and promoter hypermethylation are responsible for the underexpression of DLC1 not only in HCC but in various cancer cells and tissues (Wong et al., 2003; Durkin et al., 2007a). DLC1beta and gamma share the same promoter region and are formed by alternative splicing at the 3 end. One potential AP-1 site has been reported to be localized at -134 position in DLC1beta and gamma promoter region. The absence of CpG island in DLC1beta and gamma promoter region suggests that their expressions are less likely to be transcriptionally regulated by epigenetic alterations.

Protein size:
DLC1alpha: 1091 amino acids; 122 kDa,
DLC1beta: 1528 amino acids,
DLC1gamma: 498 amino acids.

DLC1alpha and beta are structurally similar containing SAM, RhoGAP and START domains. DLC1alpha encodes a 1091-amino acid protein with a predicted molecular weight of 122 kDa. Comparing with DLC1alpha, DLC1beta possesses a longer N-terminal region. It encodes a protein of 1529 amino acids. DLC1gamma is the smallest isoform of DLC1 with only 498 amino acids and has no functional domains. It shares the N-terminal region with DLC1beta isoform.

Functional domains:

SAM (Sterile Alpha Motif)
Interpro: IPR001660; SAM
Pfam: PF07647; SAM_2
PROSITE: PS50105; SAM_DOMAIN

RhoGAP (RhoGTPase Activating Protein)
Interpro: IPR000198; RhoGAP
Pfam: PF00620; RhoGAP
PROSITE: PS50238; RHOGAP
SMART: SM00324; RhoGAP

START (STeroidogenic Acute Regulatory related lipid Transfer)
Interpro: IPR002913; START_lipid_bd
Pfam: PF01852; START
PROSITE: PS50848; START
SMART: SM00234; START

Northern blot analysis showed that DLC1 is ubiquitously expressed in normal human tissues with high abundance in lung and ovary (Yuan et al., 1998). Quantitative PCR analysis showed that DLC1alpha is the dominant isoform in all human tissues examined, except in heart tissue whereas DLC1beta is the most abundant isoform in the heart. Among all the normal tissues examined, DLC1alpha is relatively abundant in the lung and ovary. Besides, it is moderately expressed in the thyroid, spleen, intestine and kidney, and least expressed in the adrenal gland, liver and pancreas. DLC1gamma is rarely detectable in tissues. Although DLC1 was first identified as a putative tumor suppressor gene in HCC, accumulating studies have supported that DLC1 is involved in various human cancers. Frequent underexpression of DLC1 has been detected in a wide range of tumor tissues and cancer cell lines. Both genomic deletions and epigenetic silencing are responsible for the underexpression of DLC1.

Subcellular localization of ectopically expressed DLC1 was first reported to be present in the cytoplasm of HCC cell lines, Focus and 7703K (Zhou et al., 2003). DLC1 was later reported to be colocalized with vinculin when expressed in cells (Yam et al., 2006). DLC1 has been reported to interact with tensin proteins at focal adhesions. Focal adhesion localization is crucial to the tumor suppression activity of DLC1 (Liao et al., 2007; Qian et al., 2007; Chan et al., 2009). It is also intriguing to note the presence of nuclear localization signal (NLS) and nuclear export sequences (NES) in DLC1. It is reported that the presence of a basic residues rich motif in DLC1 resembling the NLS is responsible for the nucleocytoplasmic shuttling of DLC1 (Yuan et al., 2007). It has been shown that activated protein kinase C (PKC) and protein kinase D (PKD) stimulate the association between DLC1 and 14-3-3 proteins. The enhanced association blocks DLC1 nucleocytoplasmic shuttling and inhibits the RhoGAP activity of DLC1 (Scholz et al., 2009).

Since the identification of DLC1 in 1998, accumulating studies have well characterized DLC1 as a bona fide tumor suppressor not only in HCC, but in diverse human cancers (Liao and Lo, 2008). DLC1 encodes a protein containing three major functional domains, namely, sterile alpha motif (SAM) at the N-terminus, and RhoGAP and steroidogenic acute regulatory (StAR)-related lipid transfer (START) domains at the C-terminus. RhoGAP activity has been demonstrated to be critical for the biological activities of DLC1. It possesses selective activity towards RhoA, RhoB and RhoC and less towards CDC42 but not Rac1 (Wong et al., 2003; Healy et al., 2008). Abrogation of Rho/ROCK-mediated cytoskeleton reorganization by DLC1 has been shown to be RhoGAP dependent (Wong et al., 2008). DLC1 RhoGAP defective mutant failed to inhibit stress fiber formation in HCC cells (Wong et al., 2005).
The tumor and metastasis suppressive effects of DLC1 have been well characterized by ectopic expression of DLC1 in various cancer cell lines. Extensive studies have shown that the RhoGAP activity is crucial to the biological activities of DLC1. Introduction of DLC1 into cancer cell lines has been shown to suppress cell proliferation (Ng et al., 2000), inhibit cell migration and invasion (Wong et al., 2005; Qian et al., 2007), and induce apoptosis (Zhou et al., 2004). Moreover, restoration of DLC1 expression has been shown to inhibit metastasis of cancer cells in mouse model (Goodison et al., 2005). Functional data about the loss of DLC1 in HCC tumorigenesis was first demonstrated in a mouse model using a liver-specific, short-hairpin RNA-mediated DLC1 knockdown approach (Xue at al., 2008). In p53 null; c-myc-transduced mouse hepatoblasts, RNAi knockdown of DLC1 could promote in vivo tumorigenicity of cells. Apart from RhoGAP activity, proper focal adhesion localization and interaction with tensin proteins have been demonstrated to play important roles in the biological activities of DLC1 (Yam et al., 2006; Liao et al., 2007; Qian et al., 2007; Chan et al., 2009). Other proteins including caveolin-1 (Yam et al., 2006), p120RasGAP (Yang et al., 2009) and 14-3-3 (Scholz et al., 2009) protein have also been identified as interacting partners of DLC1 with functional implications. Using knockout mouse model, DLC1 has been shown to be crucial to the early embryonic development of mouse (Durkin et al., 2005). DLC1^-/- embryos are embryonic lethal while DLC1^+/- mouse is phenotypically normal. Mouse embryonic fibroblasts isolated from DLC1 deficient mice displayed altered cytoskeletal organization and focal adhesions.

Regulation:
DLC1 is widely expressed in normal human tissues, but it is frequently underexpressed in HCC and other cancers. Heterozygous deletion and promoter hypermethylation of DLC1 are commonly found in about 30-50% of cases in various human cancers (Yuan et al., 2003a). Although DLC1 expression and activity have been well documented to be regulated at the transcriptional level, recent studies about the regulation of RhoGAP activity, interacting potentials and subcellular localization of DLC1 have pointed to an essential regulatory role by the central region of DLC1. In the central focal adhesion targeting region of DLC1, somatic mutations of DLC1 have been first detected in human prostate cancers (Liao et al., 2008). These mutations impaired the RhoGAP activity of DLC1. Crucial residues in the central region have also been shown to be responsible for proper focal adhesion localization and interacting with tensin proteins (Chan et al., 2009; Liao et al., 2007). Mutation at these crucial residues caused DLC1 to lose its focal adhesion localization and tumor suppressive activity. More importantly, the central region has been subjected to post-translational modifications. Scholz et al. have suggested that PKD-mediated DLC1 phosphorylation stimulates the association between DLC1 and 14-3-3 proteins. Enhanced association blocks DLC1 nucleocytoplasmic shuttling and inhibits the RhoGAP activity of DLC1 (Scholz et al., 2009). Moreover, identification of rat homolog of DLC1, p122RhoGAP as the substrate of Akt has provided insights into other potential regulatory pathways of DLC1 (Hers et al., 2006). However, the functional significance Akt phosphorylation in p122RhoGAP and its relevance in human DLC1 have not been investigated.

DLC family members (DLC1, DLC2 and DLC3) are structurally conserved with high sequence homology (Durkin et al., 2007b). DLC2 localizes at human chromosome 13q12.3 while DLC3 maps to human chromosome Xq13. DLC1 shares 58% and 44% amino acid identities with DLC2 and DLC3 respectively.

Somatic mutations of DLC1 have been reported to be rarely found in HCC, colorectal and ovarian cancers (Wilson et al., 2000; Park et al., 2003). Until recently, DLC1 mutations have been reported in colorectal and prostate cancers (Liao et al., 2008). Mutational analysis of human genomic and cDNA has been performed in nucleotide sequence encoding the focal adhesion targeting region (201-500 amino acids) of DLC1.

(Reference sequence: NM_006094)

Colorectal cancer (N=37)
1243 G->T (S308I) - (1/37) 2.7%
1279 G->T (S320I) - (1/37) 2.7%
1333 AC->TA (Y338L) - (1/37) 2.7%
1336 T->A (L339*) - (1/37) 2.7%

Prostate cancer (N=28)
1189 C->T (P290L) - (2/28) 7.1%
1222 C->A (T301K) - (1/28) 3.6%
1243 G->T (S308I) - (3/28) 10.7%
1249 C->A (S310*) - (1/28) 3.6%

The nonsense mutations (S310* and L339*) resulted in truncated DLC1 proteins with the loss of intact focal adhesion targeting region, RhoGAP and START domains. These truncated proteins are nonfunctional. Colony formation assay revealed that both T301K and S308I mutants displayed significant reduction in growth suppression activities. In addition, RhoGAP activity was downregulated in the two mutants as well.