**2.5 LAPTM family**

10 Hepatocellular Carcinoma – Basic Research

In addition, over-expressions of LAPTM4B mRNA and LAPTM4B-35 protein have also been found in other solid cancers. Kasper et al. (2005) reported that LAPTM4B mRNA tested by Northern Blot is over-expressed in 88% of lung cancer, 50.9% of breast cancers, 67% of colon cancers, 68% of uterus cancers, and 37% of gastric cancers. Peng et al. (2005) reported that LAPTM4B-35 protein as evaluated by immuno-histochemistry (IHC) is highly expressed in lung cancer, stomach cancer, colon cancer and breast cancer. Subsequently, the frequency of over-expression of LAPTM4B-35 in more cases of various cancers was determined, and was found in 76 % of gallbladder cancers (Zhou et al., 2007), 72 % of cholangiocarcinomas (Zhou et al.,. 2008), 63.5% of ovarian cancers (Yang et al., 2008; Yin et al., 2010), 72.57% of cervical cancers (Meng et al., 2010a) and 70.91% of endometrial cancers (Meng, et al., 2010b). However, the mechanism for over-expression of LAPTM4B in HCC and other cancers has not as yet been completely elucidated. Mutation and demethylation of *LAPTM4B* gene has not been found. Nevertheless, it has been reported in a large number of articles that the chromosome 8q region harboring *LAPTM4B* gene is amplified as shown by fluorescence in situ hybridization (FISH) or gained as shown by comparative genomic hybridization (CGH) in both hepatoblastoma and hepatocellular carcinoma (Buendia et al., 2002; Longerichet al., 2011; Marchio et al., 1997). More precisely, it has been recently reported that chromosome 8q 22 where the *LAPTM4B* gene localizes is amplified or gained in breast cancer (Hu et al., 2009; Y. Li. et al., 2010). Therefore, gene amplification may be the cytogenetic basis of LAPTM4B over-expression. However, the genomic DNA copy number alterations of most genes in general do not appear to parallel corresponding transcriptional expression (Huang et al., 2006). It is reasonable to propose that transcriptional up-regulation by transcription factors and/or microRNAs may also contribute to LAPTM4B-35 over-expression in cancers.

**2.4** *LAPTM4B* **alleles and their significance in susceptibility for hepatocarcinogenesis**  Two alleles of the *LAPTM4B* gene have been identified in our laboratory and designated *LAPTM4B \*1* and *\*2*. Allele *\*2* differs from allele *\*1* in that it contains an extra tandemly arranged 19-bp (gcttggagctccagcagct) sequence at the 5'UTR in the first exon. A PCR-based method was established for genotyping of this gene using the primers 5' GCCGACTAGGGGACTGGCGGA 3' and 5' CGAGAGCTCCGAGCTTCTGCC 3' to amplify the partial sequence of exon 1, and using genomic DNA as the template. To investigate the relationship between the allelic variants of *LAPTM4B* and the susceptibility to HCC or esophageal squamous cell carcinoma (ESC), patients with HCC or ESC, and two control groups of normal individuals from the corresponding regions were analyzed. Significant differences in the frequency of genotype *LAPTM4B\*2/2* were found in patients with HCC (17.4%) as compared with the controls (10.2%) (p<0.05), indicating that individuals with the *\*2/\*2* genotype are more susceptible to HCC than *\*1/\*1* and \**1/\*2* individuals (Table 2). However, no difference was observed in the frequencies of *LAPTM4B* genotypes in patients with ESC as compared with corresponding controls. These results suggest that allele *\*2* may be associated specifically with susceptibility to HCC. In conclusion, our data suggest that *LAPTM4B\*2/\*2* is associated with susceptibility to HCC and the *LAPTM4B* genotype provides a new means for screening for people who are susceptible to primary hepatocellular carcinoma. This may be of importance for the assessment and prevention of developing hepatocellular carcinoma in high risk populations, in particular for the patients with liver cirrhosis of small liver nodule from HBV and HCV chronic infection (Shao et al.,

A more in-depth analysis will be required to clarify these points.

In addition to LAPTM4B, LAPTM4A (Hogue et al., 1996) and LAPTM5 (Adra et al., 1996) are also members of lysosome-associated protein transmembrane (LAPTM) family. LAPTM4A (27kDa) shows a 46% homology with LAPTM4B in amino acid sequences. In comparison with LAPTM4B-35 containing 317 amino acid residues, LAPTM4A containing 233 amino acid residues and LAPTM5 containing 262 amino acid residues both are of the Nterminus-truncated molecules of LAPTM family. The three members in the LAPTM family all localize at late endosomes and lysosomes, and play an important role in lysosomal function**,** including transporting structurally unrelated amphiphilic molecules between cytosol and lysosomes, and are involved in autophagocytosis. Moreover, LAPTM4B, LAPTM4A and LAPTM5 interact and co-localize with mucolipin 1, which is a lysosomal ion channel that belongs to the transient receptor potential (TRP) superfamily and their loss-offunction mutations result in mucolipidosis type IV (MLIV), a lysosomal storage disorder characterized by severe mental and psychomotor retardation (Vergarajauregui et al., 2011). LAPTM4A is involved in the subcellular compartmentalization of diverse hydrophobic small molecules and contributes to the inherent drug sensitivity or resistance of the mammalian cell (Hogue et al., 1999). The *LAPTM5* gene maps to chromosome 1p34 and is expressed mainly in hematopoietic cells and immune cells (Adra et al., 1996). LAPTM5 protein physically interacts with the B cell receptor (BCR) complex and promotes its degradation in the lysosomal compartment in mouse B cells, and thus negatively regulates cell surface BCR levels and B cell activation (Ouchida, 2010). The expression of LAPTM5 gene is usually down-regulated through DNA methylation in a neuroblastoma cell-specific manner, while over-expression of this gene may induce spontaneous regression of neuroblastomas. It is believed that caspase-independent lysosomal cell death due to lysosomal destabilization resulted from LAPTM5 up-regulation is closely associated with the spontaneous regression (Inoue et al., 2009).

LAPTM4B: A Novel Diagnostic Biomarker and Therapeutic Target for Hepatocellular Carcinoma 13

within an 80 day period following inoculation. Half of the tumor masses generated from LAPTM4B-overexpressing NIH3T3-AE cells showed growth and were histochemically identified as malignant fibrosarcoma; the other half of these masses regressed and were finally identified as liquid lymphoid tissue. Further, the L02 cell line which originated from normal human liver was then used to generate a LAPTM4B-35 over-expressing cell model by infection with replication deficient adenovirus Ad-AE containing LAPTM4B-35 full length cDNA (Lily et al., 2011). Inoculation of LAPTM4B-35 over-expressing L02-AE cells can result in rapidly growing carcinoma xenografts in 100% (6/6) of inoculated sites in the left axilla of nude mice (Figure 7a and 7b) and shorten their live span (Figure 7d) , as

Fig. 7. Upregulation of LAPTM4B-35 promotes tumorigenesis in nude mice. (a) Xenograft tumors formed from L02-Ad-AE cells, in which LAPTM4B-35 is overexpressing, and tumors formed from L02-Ad-Null cells which are indicated by white arrows. (b) Tumor growth curves from L02-Ad-AE cells and L02-Ad-Null cells. (c) Top panel: H&E staining of tumor formed from L02-Ad-Null cells (left) and L02-Ad-AE cells (right). Black arrows indicate the cancer cells in blood vessel. Bottom panel: immunohistochemical evaluation of LAPTM4B-35 expression in xenografts. Tumors derived from L02-Ad-AE cells showed high expression of LAPTM4B-35 (right), but tumors derived from L02-Ad-Null cells showed very low expression of LAPTM4B-35 (left). An anti-LAPTM4B-N10 pAb, which specifically reacts with LAPTM4B-35, was used for IHC (original magnification 200X). (d) Kaplan–Meier survival curves plotted with SPSS 16.0 (n=6). LAPTM4B-35 up-regulation shortened the live span of mice challenged with L02-Ad-AE cells, as compared to the control group infected by Ad-null, the empty Ad vectors. P=0.049 L02-Ad-AE versus L02-Ad-Null. (Lily et al., 2011).

Notably, LAPTM family shows similarity in some functional characteristics to "tetraspanin" family, but difference in the structural characteristics, including lacking some conserved amino acid residues and having a smaller EC2 domain. (Maecker et al., 1997; Martin, 2005).

Evolutionarily, *LAPTM* is an ancient and conserved gene family in mammalian species as well as in lower eukaryotic organisms, including zebrafish (Danio rerio) and Drosophila. The phylogenic tree shown in Figure 6 is constructed according to multiple sequence alignment results. It shows that *LAPTM4B* and the other two genes in the *LAPTM* family, *LAPTM4A* and *LAPTM5*, are distributed in clusters. Human *LAPTM4B* shows the closest similarity to Bos Taurus, and also shows some similarity to its zebrafish homolog. In addition, *LAPTM4B* is more related to *LAPTM4A* than *LAPTM5*.

Fig. 6. The evolutionary lineage tree for LAPTM family (This figure was prepared by Shuang Shi)
