**LAPTM4B Targeting as Potential Therapy for Hepatocellular Carcinoma**

Rou Li Zhou, Mao Jin Li, Xuan Hui Wei, Hua Yang, Yi Shan, Ly Li and Xin Rong Liu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61345

#### **Abstract**

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HCC is one of the most common cancers worldwide with high prevalence, recurrence, and lethality. The curative rate is not satisfactory. *LAPTM4B* is a novel driver gene of HCC first indentified by our group. It is over-expressed in 87.3% of HCC. The expression levels of the encoded LAPTM4B-35 protein in HCC is also over-expressed in 86.2% of HCC and shows a significant positive correlation with pathological grade, metastasis, and recurrence, and a negative correlation with postoperative overall- and cancer freesurvival of HCC patients. Moreover, HCC cells showing high expression of LAPTM4B-35 show a strong tendency to metastasize and enhanced drug resistance. Overexpression of this gene promotes tumorigenesis, faster growth of human HCC xenografts and metasta‐ sis in nude mice, and leads to anti-apoptosis, deregulation of proliferation, enhancement of migration and invasion, as well as multi-drug resistance. In addition, overexpression of LAPTM4B-35 leads to accumulation of a number of oncoproteins and to down-regula‐ tion of a number of tumor suppressing proteins. By contrary, knockdown of endogenous LAPTM4B-35 via RNAi results in remarkable inhibition of xenograft growth and metasta‐ sis of human HCC in nude mice. Also, RNAi knockdown of LAPTN4B-35 can reverse the cellular and molecular malignant phenotypes noted above.

Therefore, it is suggested that to down-regulate over-expression of *LAPTM4B* gene and LAPTM4B-35 in HCC cells may provide novel strategy for HCC treatment. Moreover, the extensive effects caused by LAPTM4B-35 overexpression are based on its critical function in signaling network. Overexpression of LAPTM4B-35 activates at least 4 signaling path‐ ways that are commonly known to be associated with tumorigenesis. Taken together, it is suggests that *LAPTM4B* is a HCC driver gene and LAPTM4B-35 is a key protein which functions in the upstream of cancer-associated signaling network and plays a critical role in tumorigenesis, progression, metastasis, multi-drug resistance and recurrence. There‐ fore, it may be worth considering the *LAPTM4B* gene and the LAPTM4B-35 protein a novel target in cancer therapy.

In recent years, we identified small chemicals that target LAPTM4B-35 for inhibiting HCC growth and metastasis. We screened 1697 chemicals and found ethylglyoxal bisthio‐

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

semicarbazon (ETS) has effective anti-HCC activity probably via targeting LAPTM4B-35. Bel-7402 and HepG2 cell lines that highly express LAPTM4B-35 and a primarily cell line from naturally abortioned human fetal liver were used as the cell models and a control, respectively. Cell survival curve and apoptosis examination in vitro, and HCC xenograft growth and metastases in nude mice were measured to confirm the anti-HCC efficacy in vivo. Western blot, Co-IP, cDNA chips and RNAi were applied for mechanism study. The results showed that ETS can kill HCC cells but not human fetal liver cells *in vitro*, and also attenuate xenograft growth and metastasis of HCC and extend the life span of mice with HCC *in vivo*. When the endogenious over-expression of LAPTM4B-35 was knock‐ down by RNAi, the killing efficacy of ETS on HepG2 cells was significantly decreased. Also ETS inhibited the phosphorylation of LAPTM4B-35 Tyr285, which involves in activa‐ tion of PI3K/Akt signaling pathway induced by LAPTM4B-35 over-expression. In addi‐ tion, all of the molecular alterations in HepG2 cells induced by LAPTM4B-35 overexpression can be reversed by ETS, including significantly decrease of c-Myc, Bcl-2 and phosphorylated Akt, but increase of Bax and phosphorylated p53. Accordingly, apoptosis was induced by ETS, and a number of pro-apoptotic genes were upregulated, while antiapoptotic genes were downregulated. It is thus suggested that ETS may be a potential promising drug candidate for treatment of HCC by targeting LAPTM4B-35 protein.

In summary, our previous study demonstrated that *LAPTM4B* is a driver gene of HCC, targeting LAPTM4B may provide potential therapy for HCC. Targeting *LAPTM4B* in‐ cludes bio-targeted therapy and chemical-targeted therapy. The bio-targeted therapy may further explore aimed at inhibiting over-expression of *LAPTM4B* gene via RNAi, miRNA or antisense RNA etc, as well as at blacking the functions of LAPTM4B-35 pro‐ tein via specific antibody. The chemical-targeted therapy may further explore aimed at attenuating the over-activated signaling pathways in HCC by chemical inhibitors.

**Keywords:** LAPTM4B, Targeted HCC thrapy, ETS

### **1. Introduction**

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with high prevalence, recurrence, and lethality. The curative rate is not satisfactory. Lysosomal protein transmembrane 4 beta (*LAPTM4B*) is a novel driver gene of HCC first cloned and indentified by our group [1,2]. *LAPTM4B* maps to chromosome 8q22.1 and encodes three isoforms of glycoprotein with four transmembrane regions, two extracellular domains (EC1 and EC2), and one small intracellular loop, together with both N-terminal and C-terminal tails, which reside in the cytoplasm. Three isoforms of LAPTM4B protein were designated as LAPTM4B-40, LAPTM4B-35, and LAPTM4B-24 according to their molecular weights [2]. Interestingly, overexpression of LAPTM4B-35 and LAPTM4B-24 show antagonist functions: LAPTM4B-35 promotes oncogenesis and the malignant cellular and molecular phenotypes, but LAPTM4B-24 promotes apoptosis and autophage [2].

*LAPTM4B* mRNA is overexpressed in 87.3% (48/55) of HCC by Northern blot analysis. The expression levels of the encoded protein LAPTM4B-35 is also over-expressed in 86.2% (T/N≥1.5 in 56/65) of HCC by Western blot analysis [4] and 71.8% (51/71) of HCC by immunohisto‐ chemistry [3] and show a significant positive correlation with pathological grade, metastasis, and recurrence and a negative correlation with postoperative survival of HCC patients [3-5]. Moreover, HCC cells with a high expression of LAPTM4B-35 show a strong tendency to motivate drug resistance [6]. The over-expression of this gene promotes tumorigenesis, faster growth, and metastasis of human HCC xenografts in nude mice [5,7] and leads to antiapop‐ tosis, deregulation of proliferation, enhancement of migration and invasion, and multidrug resistance [5]. In addition, the overexpression of LAPTM4B-35 leads to the accumulation of a number of oncoproteins and downregulation of a number of tumor suppressing proteins. Conversely, knockdown of endogenous LAPTM4B-35 via RNAi results in remarkable inhibition of xenograft growth and metastasis of human HCC in nude mice [5,7]. Meanwhile, the RNAi knockdown of LAPTN4B-35 can reverse the cellular and molecular malignant phenotypes noted above [5]. It was also found in a number of solid cancers, including nonsmall cell lung cancer (NSCLC) that the level of LAPTM4B-35 expression was not only significantly higher than that in normal tissues and associated with histopathologic differen‐ tiation, lymph node metastasis, and TNM stage but also associated with microvessel density [8]. Taken together, it is suggested that *LAPTM4B* is a cancer driver gene and LAPTM4B-35 is a key oncoprotein, which are both predicted to be a diagnostic marker and a therapeutic target for cancer.

semicarbazon (ETS) has effective anti-HCC activity probably via targeting LAPTM4B-35. Bel-7402 and HepG2 cell lines that highly express LAPTM4B-35 and a primarily cell line from naturally abortioned human fetal liver were used as the cell models and a control, respectively. Cell survival curve and apoptosis examination in vitro, and HCC xenograft growth and metastases in nude mice were measured to confirm the anti-HCC efficacy in vivo. Western blot, Co-IP, cDNA chips and RNAi were applied for mechanism study. The results showed that ETS can kill HCC cells but not human fetal liver cells *in vitro*, and also attenuate xenograft growth and metastasis of HCC and extend the life span of mice with HCC *in vivo*. When the endogenious over-expression of LAPTM4B-35 was knock‐ down by RNAi, the killing efficacy of ETS on HepG2 cells was significantly decreased. Also ETS inhibited the phosphorylation of LAPTM4B-35 Tyr285, which involves in activa‐ tion of PI3K/Akt signaling pathway induced by LAPTM4B-35 over-expression. In addi‐ tion, all of the molecular alterations in HepG2 cells induced by LAPTM4B-35 overexpression can be reversed by ETS, including significantly decrease of c-Myc, Bcl-2 and phosphorylated Akt, but increase of Bax and phosphorylated p53. Accordingly, apoptosis was induced by ETS, and a number of pro-apoptotic genes were upregulated, while antiapoptotic genes were downregulated. It is thus suggested that ETS may be a potential promising drug candidate for treatment of HCC by targeting LAPTM4B-35 protein.

In summary, our previous study demonstrated that *LAPTM4B* is a driver gene of HCC, targeting LAPTM4B may provide potential therapy for HCC. Targeting *LAPTM4B* in‐ cludes bio-targeted therapy and chemical-targeted therapy. The bio-targeted therapy may further explore aimed at inhibiting over-expression of *LAPTM4B* gene via RNAi, miRNA or antisense RNA etc, as well as at blacking the functions of LAPTM4B-35 pro‐ tein via specific antibody. The chemical-targeted therapy may further explore aimed at attenuating the over-activated signaling pathways in HCC by chemical inhibitors.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with high prevalence, recurrence, and lethality. The curative rate is not satisfactory. Lysosomal protein transmembrane 4 beta (*LAPTM4B*) is a novel driver gene of HCC first cloned and indentified by our group [1,2]. *LAPTM4B* maps to chromosome 8q22.1 and encodes three isoforms of glycoprotein with four transmembrane regions, two extracellular domains (EC1 and EC2), and one small intracellular loop, together with both N-terminal and C-terminal tails, which reside in the cytoplasm. Three isoforms of LAPTM4B protein were designated as LAPTM4B-40, LAPTM4B-35, and LAPTM4B-24 according to their molecular weights [2]. Interestingly, overexpression of LAPTM4B-35 and LAPTM4B-24 show antagonist functions: LAPTM4B-35 promotes oncogenesis and the malignant cellular and molecular phenotypes, but

*LAPTM4B* mRNA is overexpressed in 87.3% (48/55) of HCC by Northern blot analysis. The expression levels of the encoded protein LAPTM4B-35 is also over-expressed in 86.2% (T/N≥1.5 in 56/65) of HCC by Western blot analysis [4] and 71.8% (51/71) of HCC by immunohisto‐ chemistry [3] and show a significant positive correlation with pathological grade, metastasis,

**Keywords:** LAPTM4B, Targeted HCC thrapy, ETS

LAPTM4B-24 promotes apoptosis and autophage [2].

**1. Introduction**

148 Recent Advances in Liver Diseases and Surgery

The extensive effects caused by LAPTM4B-35 overexpression are based on its critical function on cell trafficking and signaling network. Recently, Tan et al. [9] reported that the oncoprotein LAPTM4B not only interacts with EGFR but also regulates EGFR internalization and traffick‐ ing, and thus increases the amount and enhances the functions of EGFR on cell surface. Moreover, LAPTM4B can play a kinase-independent role for EGFR in autophagy initiation [10]. We found that the over-expression of LAPTM4B-35 can activate several signaling pathways that are commonly known to be associated with oncogenesis and progression [2]. The activation of PI3K/Akt signaling pathway induced by the overexpression of LAPTM4B-35 has been demonstrated to associate with drug resistance [6]. In this paper, we further present the functions of LAPTM4B-35 on signaling and a chemical that inhibits HCC *in vitro* and *in vivo* by targeting LAPTM4B-35.
