LAPTM4B Targeting as Potential Therapy for Hepatocellular Carcinoma

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 free-survival 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 (ETS) 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 Tyr 285 , 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 anti- apoptotic 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. 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. overexpressed HCC cells, activation ERK FN/

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 knockdown by RNAi, the killing efficacy of ETS on HepG2 cells was significantly decreased. Also ETS inhibited the phosphorylation of LAPTM4B-35 Tyr 285 , which involves in activation of PI3K/Akt signaling pathway induced by LAPTM4B-35 over-expression. In addition, 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 includes bio-targeted therapy and chemical-targeted therapy. The bio-targeted therapy

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 immunohistochemistry [3] and show a significant positive correlation with pathological grade, metastasis, and recurrence and a negative correlation with postoperative survival of HCC patients [3][4][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 antiapoptosis, 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 differentiation, 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.
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 trafficking, 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.

Functions of LAPTM4B-35 involved in signaling network
Current evidence indicates that the interaction between cancer cells with their microenvironment plays key roles in oncogenesis and progression. Cancer microenvironment is composed of variant signal molecules, including solvable signal molecules (growth factors, cytokines, etc.), insolvable extracellular matrix (ECM), and variant cells nearby. Cancer cells and their microenvironment are reciprocally affected. Cancer cell proliferation, survival, and migrationare all motivated and dependent on not only solvable signal molecules but also ECM. Cancer cells accept positive or negative regulations of signal molecules from solvable factors, ECM, and other cells in their microenvironment through signal transduction pathways, which are organized as a very complicated network. In other words, cancer may be known as a disease of signaling network. Disturbances of signaling pathways and the converging network initiate at the early stage and go through the whole process of cancer development. In addition, the disturbance of signaling pathways results from oncogenic alternation in genetics and epigenetics and contributes to the molecular and cellular malignant phenotypes of cancer cells, which include disregulations of proliferation, survival/apoptosis, differentiation and metabolism, as well as enhancement of migration/invasion and multidrug resistance. Therefore, signaling pathways and the network are of importance from a therapeutic perspective because targeting them may help reverse, delay, or prevent oncogenesis. Notably, since cirrhosis is associated with hepatic regeneration after tissue damages, which are caused by hepatitis infection, toxins (for example, alcohol oraflatoxin) or metabolic influences, and is often the prerequisite of hepato-oncogenesis, it is noticed that the ECM and the ECM-related signaling pathways, that are commonly alternated in cirrhosis and HCC, are of very importance. Our preliminary study has indicated that LAPTM4B-35 is most likely an assembly platform or organizer for a number of signaling molecules which are integrated in the cell membranes or soluble in the cytoplasm. Overexpression of LAPTM4B-35 would therefore be expected to lead to disturbance of a wide range of signaling pathways and their networks. We found LAPTM4B-35 can interact or co-localize with a number of these signal molecules, including membrane-integrated receptors and cytoplasmic signal molecules. These membrane-integrated receptors involve the growth factor receptors of the RTK (receptor tyrosine kinase) family, such as EGFR [9][10][11] and IGF-1R (Figure 1a), and ECM receptors of the integrin family, such as α6β1 [11] and α5β1 (Figures 1d and 2). The cytoplasmic signaling molecules that can interact with LAPTM4B-35 include FAK ( Figure 2c) and PI3K p85α (Figure 3a). Given that LAPTM4B-35 is a tetra-transmembrane protein and localizes in plasma membrane and endomembranes (including lysosomes and endosomes). The interaction of LAPTM4B-35 with both RTK under the stimulation of growth factors, and integrin under ECM stimulation would be expected to integrate related signal transduction pathways triggered by growth factors and ECM components at the cell surface. It is well known that based on binding of growth factors (ligand) to their corresponding RTK receptor, Ras and ERK1/2 (MAPK family)downstream is subsequently activated [12]. At the same time, based on binding of ECM components (ligand), such as fibronectin (FN) or laminin (LN), to their corresponding integrin receptor (α5β1 or α6β1, respectively), FAK 397 is phosphorylated and activated, and may subsequently activate downstream Ras/ERK and PI3K/AKT signaling pathways [13,14]. As has been previously recognized, the RTK/Ras/ERK signaling pathway and the ECM/Integrin/FAK signaling pathway converge at Ras and/or FAK. However, we found that over expression of LAPTM4B-35 can not only dramatically activate Ras ( Figure 1b Figure 1f. AE and Mock cells are both LAPTM4B-35 overexpressed, but to different extents. These results suggest that in LAPTM4B-35 overexpressed HCC cells, activation of ERK results from both the upstream growth factor/Ras and FN/ Integrin/FAK signaling cascades. Taken together, it is reasonable to propose that overexpressed LAPTM4B-35 as a linker at the cell surface (plasma membrane) simultaneously over activates both the growth factor (EGF or IGF-1R)/RTK/Ras/ERK and the ECM (FN or LN)/ Integrin/FAK/ERK signaling pathways by interacting with growth factor receptor (RTK) and ECM receptor (integrin) under the stimulation of growth factor and ECM components (FN, LN), respectively. In other words, the growth factor/RTK/Ras/ERK and ECM/ integrin/FAK/ERK signaling pathways initiallyconverge at the plasma membrane level through overexpression of membrane-integrated LAPTM4B-35 in HCC cells, instead of at Ras and FAK in the cytoplasm in normal hepatocytes whichexpress LAPTM4B-35 and FAK at rather low level. Moreover, simultaneous overactivation of these two signaling pathways caused by LAPTM4B-35 overexpression would result in enhancement of proliferation, survival, migration and invasion of cancer cells.  We found that not only membrane-integrated receptors, but also some solvable signaling molecules in cytoplasm can interact with LAPTM4B-35, such as FAK ( Figure 2c) and PI3K p85α [6]. It is known that PI3K is a kinase which catalyzes phosphorylation of proteins andlipids. An important phosphorylated product catalyzed byPI3K is membrane-integrated PIP3 which can recruit cytoplasmic PH domain-containing proteins, including Akt and the corresponding kinases (PDK1 and PDK2) to the plasma membrane where Akt is phosphorylated by PDK1 and PDK2. Phosphorylated Akt is commonly known as a marker for PI3K/Akt signaling pathway activation. In view of the fact PI3K consists of two subunits: p110 catalytic subunit and p85α regulatory subunit. The kinase activity of p110 is normally inhibited by binding of p85α. The inhibitory effect of p85αcan be released by binding to an appropriate molecule [15]. We found that LAPTM4B-35 can interact with p85α, but not with p110 ( Figure  3a). Moreover, using site-directed mutation experiments we found that binding of LAPTM4B-35 to PI3K p85α is mediated by two motifs. One is the proline-rich motif (PPRP) in the N-terminus of LAPTM4B-35, which may bind to the SH3 domain of PI3K p85αsubunit, and the other is phosphorylated Tyr 285 in the C-terminus of LAPTM4B-35, which may bind to the SH2 domain of the PI3K p85α subunit (Figure 3b). To demonstrate this a series of HCC cell variants with highly expressed wild type and mutated LAPTM4B-35 were prepared by transfection with variant plasmids containing LAPTM4B-35 with mutation at PPRP or at Try It is known that FAK, as a functionally complicated signal molecule with Tyr kinase activity and nonkinase scaffolding function, is overexpressed in many cancers (including 60% of HCC) and involves in many aspects of tumor growth, invasion, and metastasis. Given that the phophorylation of FAK Tyr 397 is critical for trigering its Tyr kinase activity and enhancing its nonkinase scaffolding function, and is induced by binding of integrin with FN or LN. We found that the PI3K/Akt signaling pathway in LAPTM4B-35 overexpressed HCC cells can be activated by stimulation of not only serum but also fibronectin or laminin substrate (   In summary, cancer-targeted therapy currently focuses primarily on targeting key signaling molecules in one or more signaling pathways which are overactivated in a given cancer. Tetratransmembrane LAPTM4B-35 is believed to function as an assembly platform or organizer for a number of signaling molecules, which may either be integrated in the cell membranes or soluble in the cytoplasm. The LAPTM4B-35 overexpression, which occurs in more than 80% of HCC tissues, and the interactions with membrane-integrated receptors and cytoplasmic signal molecules are expected to act as an amplified assembly platform for upstream signal molecules of several signaling pathways, and leads to over activation of related signaling pathways ( Figure 5), such as growth factor/ RTK/Ras/ERK, growth factor/RTK/Ras/PI3K/Akt, ECM/integrin/FAK/ERK, ECM/integrin/FAK/PI3K/Akt, and so on. Since these signaling pathways and their networks are closely associated with malignant molecular and cellular phenotypes, including cell proliferation/differentiation and survival/apoptosis as well as migration/invasion, it is believed that over activation of these signaling pathways is linked with hepatic carcinogenesis and progression [12][13][14][15]. Collectively, our data strongly suggest that LAPTM4B-35 would be an ideal target for HCC treatment, and that LAPTM4B-targeted therapy is a promising potential therapeutic strategy for HCC which will act in down regulation of the expression of LAPTM4B-35, or act by obstructing the interaction of LAPTM4B-35 with growth factors, integrins, FAK, PI3K p85α and other LAPTM4B-35 binding signal molecules.

Small chemicals targeting LAPTM4B-35
The molecular targets for cancer therapy have expanded from angiogenesis to oncogenic signaling pathways. The target indication has shifted from advanced stage to early or inter-   [17,20].
Based on the basic characteristics: (1) LAPTM4B is a driver oncogene (2) this gene and theencoding LAPTM4B-35 protein are over expressed in more than 85% of HCC and (3) theoverexpression of LAPTM4B-35 can activate multiplesignaling pathways, we propose that LAPTM4B gene and the LAPTM4B-35 protein might bean ideal target for HCC treatment. We identified the chemicals that target LAPTM4B-35 for inhibiting HCC growth and metastasis. A total of 1697 synthetic small chemicals from Li and Liu (Pharmaceutical Institute, Chinese Academy of Medical Sciences) were screened. Among these chemicals, ethylglyoxal bisthiosemicarbazone (ETS) was found to have effective activity for the inhibition of growth and metastasis of human HCC cells in vitro and in vivo probably via targeting LAPTM4B-35 [18].
Three HCC cell lines (Bel-74402, HepG2, and HLE) from human HCC and a cell line from naturally aborted human fetal were used as the cell models and a control, respectively. Cell survival curve and apoptosis analysis in vitro and HCC xenograft growth and metastasis in nude mice were evaluated to confirm the inhibitory efficacy in vivo. Western blot, Co-IP, cDNA chips, and RNAi were applied for exploration on mechanism.
We found that ETS can inhibit cell growth of variant HCC cell lines in a dose-dependent manner shown by cell growth curve in vitro (Figure 6a, 6b, and 6d). The  (Figure 6b). Moreover, the killing efficacy of ETS was confirmed from two aspects. First, after ETS treatment at a concentration of 1.25 μM for 72 h, HepG2 cells were cultured in a ETS-free medium at 37°C for as long as 12 days. As a result, when compared with 6 × 10 3 cells seeded in a well at the beginning, only a few colonies appeared after the12 days ETS-free culture, indicating that the vast majority of HepG2 cells were killed by ETS. Second, the significant killing efficacy of ETS on HepG2 cells was further confirmed by Calcein-AM/EthD-1 fluorescence double staining in a time-dependent manner (Figure 7b). The time-dependent growth inhibition was also shown by growth curves of HepG2 cells in vitro (Figure 7a) and HCC xenograft in vivo (Figure 8a). ETS also shows significant effect on the inhibition of HCC growth and metastasis in vivo.
Human HCC BEL-7402 cells were subcutaneously inoculated, and then ETS was administered either by intratumor injection or intraperitoneal injection. Both ways can inhibit the HCC xenograft growth. The effect of ETS on attenuation of growth and metastasis of human HCC xenograft in nude mice is shown in Table 1, as well as Figure 8(a) and 8(b). At the same time, the mice treated by ETS were less lost their body weight than that treated by mitomycin and cisplatin. As a matter of fact, the acute toxicity test indicated that ETS had little poison on mice.
There is no death of mice in the 1000mg/kg, 464mg/kg, and control groups. Of the 10 mice per group, all died in the 4640mg/kg group, and 4 mice died in the 2150mg/kg group. The LD 50 of ETS was 2329.9 mg/kg, with a 95% dependable limit of 1846.7-2939.0 mg/kg.
In addition, a murine HCC H22 cell line was applied to study the effect of ETS on the life span of mice with ascetic HCC. A dose-dependent prolongation of life span was observed as shown in Figure 8(c).
To illustrate the mechanism for killing HCC cells of ETS, apoptosis was studied at cellular, molecular, and gene levels.   9a). Notably, the phosphorylation of p53 protein is also increasing, suggesting that ETS might stabilize p53 protein, the key apoptosis regulator. Western blot analysis also showed that the key effecter molecule of apoptosis pathway, caspase 3, was activated from procaspase into cleaved caspase by ETS in a time-dependent manner (Figure 9c). At the same time, cDNA array analysis showed that a large number of proapoptotic genes were up-regulated and a large number of antiapoptotic genes were down-regulated by ETS treatment (Figure 9d).
Based on LAPTM4B-35 overexpression in HCC can up-regulate a number of oncogenes that promote cell proliferation and/or resist apoptosis, the effects of ETS on the expression of oncoproteins were detected. We found that all the molecular alterations in HepG2 cells induced by LAPTM4B-35 overexpression can be reversed by ETS (Figures 9-11), such as significant decrease of c-Myc (Figure 9b), cyclinD1, and Bcl-2 ( Figure 9a) but increase of Bax and phosphorylated p53 (Figure 9a).
It is well known that PI3K/Akt signaling pathway plays a key role in antiapoptosis and cell survival in a large number of cancers and thus is considered as a target for cancer therapy [20]. We have found that the PI3K/Akt/GSK3β signaling pathway is overactivated by LAPTM4B-35 overexpression [5,6]. The effect of ETS on PI3K/Akt signaling was detected.We found that the phosphorylated Akt (Akt-p) is significantly reduced in the ETStreated HCC cells either in the presence or absence of serum stimulation (Figure 10a). Then the mechanism was explored. Co-IP and Western blot analyses showed that ETS significantly decreased the phosphorylation of LAPTM4B-35 Tyr 285 in C-terminus of LAPTM4B-35 ( Figure 10b) and therefore the activation of PI3K/Akt signaling pathway is minimized via reducing interaction of LAPTM4B-35 and PI3K p85α ( Figure 12).
In summary, our previous study demonstrated that LAPTM4B is a driver gene of HCC, and LAPTM4B-35 targeting may provide potential therapy for HCC. To target LAPTM4B for cancer therapy includes bio-targeted therapy and chemical-targeted therapy. The bio-targeted therapy may further explore aimed at inhibiting the overexpression of LAPTM4B gene via RNAi, miRNA, or antisense RNA, etc., as well as at blocking the functions of LAPTM4B-35 protein via specific antibody. The chemical-targeted therapy may further explore aimed at attenuating the overactivated signaling pathways by chemical inhibitors and thus inhibiting proliferation and inducing apoptosis. More signaling pathways and more complicated signaling network are supposed to be involved in deregulation induced by LAPTM4B-35 overexpression in cancer. Thus, the mechanism of ETS for targeting LAPTM4B-35 may be more complicated.
-20 -   HepG2 cells were first serum-starved for 16 h, then serum and ETS or PBS (control) were added for 15min. The cell lysate was first precipitated by anti-LAPTM4B-N10-pAb, which reacts with LAPTM4B-35. After absorption by protein G/A agarose beads, the precipitant was subjected to Western blot analysis with antiphosphorylated Tyr-mAb. The profile shows that compared with the control, the phosphorylated LAPTM4B-35 is attenuated by ETS treatment in either presence or absence of serum stimulation.

Conclusion
Given that LAPTM4B is a driver gene of HCC and the encoding LAPTM4B-35 protein is overexpressed in HCC and contributes to the cellular and molecular malignant phenotypes [2], the study on molecular mechanism reveals that the overexpression in HCC of the membrane integrated LAPTM4B-35 functions as an amplified assembly platform or organizer of related signaling molecules that are either integrated in cell membranes or solvable in cytoplasm, and thus activates several signaling pathways, such as growth factor/ RTK/Ras/ERK (MAPK), growth factor/RTK/Ras/PI3K/Akt, ECM/integrin/FAK/ERK (MAPK) or ECM/integrin/FAK/ PI3K/Akt, etc. Therefore, it is worth considering the LAPTM4B gene and the LAPTM4B-35 protein as novel targets in HCC therapy. A small chemical (ETS) can inhibit HCC cell growth and induce apoptosis in vitro, and inhibit growth and metastasis of human HCC xenograft in vivo. Notably, ETS can reverse the molecular alterations, that are induced by LAPTM4B-35 overexpression and involved in promotion of proliferation and survival of cancer cells. Moreover, ETS inhibits the phosphorylation of LAPTM4B-35 Tyr 285 , a key motif forbinding to PI3K p85α regulatory subunit,, and thus inhibits the PI3K/Akt signaling pathway. Taken together, developing strategies for LAPTM4B-35 targeting can be a potential treatment for hepatocellular carcinoma therapy.