**3. 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‐ mediate stages of cancer. Agents targeting EGFR, FGFR, PI3K/Akt/mTOR, TGF-β, c-Met, MEK, IGF signaling, FAK and histone deacetylase have been actively explored [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 IC50 of ETS inhibition varies for variant HCC cell lines, such as HepG2 (0.9 μmol/L), Bel-7402 (0.7 μmol/L), HLE (1.1 μmol/L), and H22 (1.6 μmol/L). Convesely, ETS cannot affect the survival of human fetal liver cells even if the concentration of ETS is increasing to as high as 200 times of that used for HCC cells*.* Notably, both Bel-7402 and HepG2 cells express LAPTM4B-35 at very high level and are most sensitive to ETS; HLE cells express LAPTM4B-35 at relatively low level [19] and are less sensitive to ETS. However, the fetal liver cells that express LAPTM4B-35 at a low level are not sensitive to ETS. Accordinly, when the endogenous overexpression of LAPTM4B-35 was knocked down by RNAi through shRNA transfection, the inhibitory effect of ETS on HepG2 cells was significantly decreased (Figure 6d). Figure 6c demonstrates the killing efficacy of ETS to HepG2 cell as shown by fluorescently double stained with Calcein-AM (1 μmol/L) and EthD-1 (2 μmol/L). Cells emitting green fluorescence were alive cells merely stained by Calcein-AM. Cells emitting red fluorescence were dead cells or apoptotic cells merely stained by EthD-1. Collectively, It is suggested that the inhibitory/killing efficacy of ETS on HCC cells depends on the high expression of LAPTM4B-35. At the same time, the effect of ETS on HepG2 cells was more effective than cisplatin (IC50: 7.5 μmol/L), doxorubicin (IC50: 7.6 μmol/L), mitomycin (IC50: 5.8 μmol/L), and 5-fluorouracil (IC50: >200 μmol/L) *in vitro* (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 × 103 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).

mediate stages of cancer. Agents targeting EGFR, FGFR, PI3K/Akt/mTOR, TGF-β, c-Met, MEK,

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

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 IC50 of ETS inhibition varies for variant HCC cell lines, such as HepG2 (0.9 μmol/L), Bel-7402 (0.7 μmol/L), HLE (1.1 μmol/L), and H22 (1.6 μmol/L). Convesely, ETS cannot affect the survival of human fetal liver cells even if the concentration of ETS is increasing to as high as 200 times of that used for HCC cells*.* Notably, both Bel-7402 and HepG2 cells express LAPTM4B-35 at very high level and are most sensitive to ETS; HLE cells express LAPTM4B-35 at relatively low level [19] and are less sensitive to ETS. However, the fetal liver cells that express LAPTM4B-35 at a low level are not sensitive to ETS. Accordinly, when the endogenous overexpression of LAPTM4B-35 was knocked down by RNAi through shRNA transfection, the inhibitory effect of ETS on HepG2 cells was significantly decreased (Figure 6d). Figure 6c demonstrates the killing efficacy of ETS to HepG2 cell as shown by fluorescently double stained with Calcein-AM (1 μmol/L) and EthD-1 (2 μmol/L). Cells emitting green fluorescence were alive cells merely stained by Calcein-AM. Cells emitting red fluorescence were dead cells or apoptotic cells merely stained by EthD-1. Collectively, It is suggested that the inhibitory/killing efficacy of ETS on HCC cells depends on the high expression of LAPTM4B-35. At the same time, the effect of ETS on HepG2 cells was more effective than cisplatin (IC50: 7.5 μmol/L), doxorubicin (IC50: 7.6 μmol/L), mitomycin (IC50: 5.8 μmol/L), and 5-fluorouracil (IC50: >200 μmol/L) *in vitro* (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

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).

cells seeded in a well at the

chips, and RNAi were applied for exploration on mechanism.

for as long as 12 days. As a result, when compared with 6 × 103

IGF signaling, FAK and histone deacetylase have been actively explored [17,20].

158 Recent Advances in Liver Diseases and Surgery

**Figure 6.** Inhibitory and killing efficiency of ETS on HCC cells. *(a)* Cancer cells of variant lines were incubated in the absence or presence of ETS at indicated concentrations for 48 h. *(b)* HepG2 cells were incubated in the absence or pres‐ ence of variant drugs at indicated concentrations for 48 h. *(c)* The cells were fluorescently double-stained with Calcein-AM (1 μmol/L) and EthD-1 (2 μmol/L) at 37℃ for 30 min and then surveyed under fluorescence microscope. Cells emitting green fluorescence were alive cells which were merely stained by Calcein-AM. Cells emitting red fluorescence were dead cells or apoptotic cells which merely stained by EthD-1. Upper panel: HepG2 HCC cells were treated by ETS at a concentration of 2 μmol/L for 48 h. The vast majority of HepG2 cells were killed by ETS. Lower panel: human fetal liver cells were treated by ETS at a concentration of 25 μmol/L for 48 h. None of fetal liver cells were killed by ETS. *(d)* HepG2 cell line was transfected by LAPTM4B-shRNA or Mock. The transfected HepG2 cells by LAPTM4BshRNA (RNAi) or LAPTM4B-Mock plasmids and the parent HepG2 cells were treated by ETS at indicated concentra‐ tions for 48 h. The LAPTM4B-35 silenced HepG2 cells showed less sensitive to ETS.The cell survival rate (%) of growth curves was calculated according to ratio of viable cells number determined by acid phosphatase assay (APA) before and after treatment.

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

concentrations for 48 h. The LAPTM4B-35 silenced HepG2 cells showed less sensitive to

cells number determined by acid phosphatase assay (APA) before and after treatment.

Figure 7. The time- and dose-dependent inhibition and killing of ETS on HepG2 cells. *(a)* HepG2 cells were treated with ETS at variant concentrations or for variant hours. The number of viable cells was determined by ASA. This figure shows that the effect of ETS on inhibiting HCC cell growth is dose and time dependent. *(b)* HepG2 cells were treated as **Figure 7.** The time- and dose-dependent inhibition and killing of ETS on HepG2 cells. (*a)* HepG2 cells were treated with ETS at variant concentrations or for variant hours. The number of viable cells was determined by ASA. This fig‐ ure shows that the effect of ETS on inhibiting HCC cell growth is dose and time dependent. *(b)* HepG2 cells were treat‐ ed as Figure 6(c). Upper panel: with ETS (2 μM) for indicated incubation time. Lower panel: without ETS for comparable incubation time as a control. This figure shows that the effect of ETS on killing HCC cells is time-depend‐ ent.

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 LD50 of ETS was 2329.9 mg/kg, with a 95% dependable limit of 1846.7-2939.0 mg/kg. Figure 6(c). Upper panel: with ETS (2 μM) for indicated incubation time. Lower panel: without ETS for comparable incubation time as a control. This figure shows that the effect of ETS on killing HCC cells is time-dependent.

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). ETS also shows significant effect on the inhibition of HCC growth and metastasis *in vivo*. Human HCC BEL-7402 cells were subcutaneously inoculated,

To illustrate the mechanism for killing HCC cells of ETS, apoptosis was studied at cellular, molecular, and gene levels. Flow cytometry showed that ETS (2μmol/L) can induce apoptosis of HepG2 cells in a time-dependent manner, i.e., 10.1% (8 h), 15.8% (16 h), 29.1% (24 h), 63.0% (36 h), and ~100% (48 h). The apoptotic cell rate includes all apoptotic cells at early and late apoptotic phases. Western blot analysis showed that along with the prolonged time of ETS treatment, the antiapoptotic Bcl-2 is decreasing and proapoptotic Bax is increasing (Figure ‐ 18 ‐ and then ETS was administered either by intratumor injection or intraperitoneal


\*\**p* < 0.01 vs. controls.

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 LD50 of

Figure 7. The time- and dose-dependent inhibition and killing of ETS on HepG2 cells. *(a)* HepG2 cells were treated with ETS at variant concentrations or for variant hours. The number of viable cells was determined by ASA. This figure shows that the effect of ETS on inhibiting HCC cell growth is dose and time dependent. *(b)* HepG2 cells were treated as Figure 6(c). Upper panel: with ETS (2 μM) for indicated incubation time. Lower panel: without ETS for comparable incubation time as a control. This figure shows that the effect of ETS on killing HCC cells is time-dependent.

**Figure 7.** The time- and dose-dependent inhibition and killing of ETS on HepG2 cells. (*a)* HepG2 cells were treated with ETS at variant concentrations or for variant hours. The number of viable cells was determined by ASA. This fig‐ ure shows that the effect of ETS on inhibiting HCC cell growth is dose and time dependent. *(b)* HepG2 cells were treat‐ ed as Figure 6(c). Upper panel: with ETS (2 μM) for indicated incubation time. Lower panel: without ETS for comparable incubation time as a control. This figure shows that the effect of ETS on killing HCC cells is time-depend‐

concentrations for 48 h. The LAPTM4B-35 silenced HepG2 cells showed less sensitive to ETS.The cell survival rate (%) of growth curves was calculated according to ratio of viable cells number determined by acid phosphatase assay (APA) before and after treatment.

160 Recent Advances in Liver Diseases and Surgery

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

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

To illustrate the mechanism for killing HCC cells of ETS, apoptosis was studied at cellular, molecular, and gene levels. Flow cytometry showed that ETS (2μmol/L) can induce apoptosis of HepG2 cells in a time-dependent manner, i.e., 10.1% (8 h), 15.8% (16 h), 29.1% (24 h), 63.0% (36 h), and ~100% (48 h). The apoptotic cell rate includes all apoptotic cells at early and late apoptotic phases. Western blot analysis showed that along with the prolonged time of ETS treatment, the antiapoptotic Bcl-2 is decreasing and proapoptotic Bax is increasing (Figure

‐ 18 ‐

ETS was 2329.9 mg/kg, with a 95% dependable limit of 1846.7-2939.0 mg/kg.

in Figure 8(c).

ent.

**Table 1.** Inhibitory efficacy of ETS on the xenograph of human HCC in nude mice

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 detect‐ ed.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 significant‐ ly decreased the phosphorylation of LAPTM4B-35 Tyr285 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

Figure 8. Inhibitory effect of ETS on growth and metastasis of human HCC xenograft in nude mice. Human HCC Bel-7402 cells (1 × 106 ) were inoculated into each nude mice. ETS (5, 15, or 45 mg/kg), cisplatin (2.0 mg/kg), mitomycin (2.0 mg/kg), PBS (control 1), or solvent (control 2) was administered every other day for each BALB\c-nude mouse in variant group (*n* = 8), respectively, by intraperitoneal injection from day 9 when the xenograft grew out. Tumor volume was measured twice a week. The inhibitory efficacy on xenograft growth of ETS was **Figure 8.** Inhibitory effect of ETS on growth and metastasis of human HCC xenograft in nude mice. Human HCC Bel-7402 cells (1 × 106 ) were inoculated into each nude mice. ETS (5, 15, or 45 mg/kg), cisplatin (2.0 mg/kg), mitomycin (2.0 mg/kg), PBS (control 1), or solvent (control 2) was administered every other day for each BALB\c-nude mouse in variant groups (*n* = 8), respectively, by intraperitoneal injection from day 9 when the xenograft grew out. Tumor vol‐ ume was measured twice a week. The inhibitory efficacy on xenograft growth of ETS was observed to be dose-depend‐ ent as compared with the control groups of solvent and PBS. Mitomycin and cisplatin were used as the positive controls. *(a)* Tumor growth curves of human HCC xenograft in nude mice with variant treatments. *(b)* Tumor photo‐ graph of human HCC xenograft in nude mice with variant treatment for 6 weeks. Left panel: Size of human HCC xen‐ ografts in variant groups. Right panel: Number of lymph node metastases in variant groups. *(c)* The survival curves of mice with ascetic HCC in variant groups. Mouse hepatocellular carcinoma H22 cells (1 × 106 ) were inoculated into peri‐ toneal of each ICR mouse. ETS (0.5 or 1.5 mg/kg) or the solvent was intraperitoneally administered every other day for each ICR mouse in variant groups (*n* = 10). The life span showed a significant prolongation in the ETS groups in a dose-dependent manner.

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. Mitomycin and cisplatin were used as the positive controls. *(a)* Tumor growth curves of human HCC xenograft in nude mice with variant treatments. *(b)* Tumor photograph of human HCC xenograft in nude mice with variant treatment for 6 weeks. Left panel: Size of human HCC xenografts in variant groups. Right panel: Number of lymph node metastases in variant

observed to be dose-dependent as compared with the control groups of solvent and PBS.

‐ 20 ‐

LAPTM4B Targeting as Potential Therapy for Hepatocellular Carcinoma http://dx.doi.org/10.5772/61345 163

**Figure 9.** Apoptosis-related molecular alteration induced by ETS. *(a)* Western blot profiles of cyclin D1, Bcl-2, Bax, and phosphorylated p53 proteins from lysates of HepG2 cells incubated in the presence of ETS (2 μM) for indicated times, indicating that proliferation- and apoptosis-related proteins are altered by ETS in a time-dependent manner. *(b)* West‐ ern blot profile of cMyc protein from lysates of HepG2 cells incubated in the presence of ETS at indicated concentra‐ tion for indicated hours, indicating remarkable decrease of c-Myc protein by treatment of ETS in a dose- and timedependent manner. *(c)* Western blot profile of procaspase 3 and cleaved caspase 3 from lysates of HepG2 cells incubated in the presence of ETS (2 μM) for indicated times, indicating the activation of key effecter molecule in apop‐ totic pathway by ETS. *(d)* cDNA array analysis shows the up-regulated and down-regulated genes that promote and inhibit apoptosis, respectively, by treatment of ETS.

**Figure 10.** Inhibitory effects of ETS on phosphorylation of Akt and LAPTM4B-35. *(a)* Western blot profile of phos‐ phorylated Akt from lysates of HepG2 cells incubated in the absence and presence of ETS (2 μM), indicating the inhibi‐ tory effect of ETS on activation of PI3K/Akt signaling pathway under stimulation with and without serum. *(b)* Co-IP and Western blot profile shows that ETS significantly decreased the phosphorylation of Tyr of LAPTM4B-35 protein. 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 pro‐ file shows that compared with the control, the phosphorylated LAPTM4B-35 is attenuated by ETS treatment in either presence or absence of serum stimulation.

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

toneal of each ICR mouse. ETS (0.5 or 1.5 mg/kg) or the solvent was intraperitoneally administered every other day for each ICR mouse in variant groups (*n* = 10). The life span showed a significant prolongation in the ETS groups in a

human HCC xenograft in nude mice with variant treatments. *(b)* Tumor photograph of human HCC xenograft in nude mice with variant treatment for 6 weeks. Left panel: Size of human HCC xenografts in variant groups. Right panel: Number of lymph node metastases in variant

Figure 8. Inhibitory effect of ETS on growth and metastasis of human HCC xenograft in nude mice.

**Figure 8.** Inhibitory effect of ETS on growth and metastasis of human HCC xenograft in nude mice. Human HCC

(2.0 mg/kg), PBS (control 1), or solvent (control 2) was administered every other day for each BALB\c-nude mouse in variant groups (*n* = 8), respectively, by intraperitoneal injection from day 9 when the xenograft grew out. Tumor vol‐ ume was measured twice a week. The inhibitory efficacy on xenograft growth of ETS was observed to be dose-depend‐ ent as compared with the control groups of solvent and PBS. Mitomycin and cisplatin were used as the positive controls. *(a)* Tumor growth curves of human HCC xenograft in nude mice with variant treatments. *(b)* Tumor photo‐ graph of human HCC xenograft in nude mice with variant treatment for 6 weeks. Left panel: Size of human HCC xen‐ ografts in variant groups. Right panel: Number of lymph node metastases in variant groups. *(c)* The survival curves of

) were inoculated into each nude mice. ETS (5, 15, or 45 mg/kg), cisplatin (2.0 mg/kg), mitomycin

mg/kg), cisplatin (2.0 mg/kg), mitomycin (2.0 mg/kg), PBS (control 1), or solvent (control 2) was administered every other day for each BALB\c-nude mouse in variant group (*n* = 8), respectively, by intraperitoneal injection from day 9 when the xenograft grew out. Tumor volume was measured twice a week. The inhibitory efficacy on xenograft growth of ETS was observed to be dose-dependent as compared with the control groups of solvent and PBS. Mitomycin and cisplatin were used as the positive controls. *(a)* Tumor growth curves of

mice with ascetic HCC in variant groups. Mouse hepatocellular carcinoma H22 cells (1 × 106

) were inoculated into each nude mice. ETS (5, 15, or 45

) were inoculated into peri‐

‐ 20 ‐

complicated.

Human HCC Bel-7402 cells (1 × 106

162 Recent Advances in Liver Diseases and Surgery

Bel-7402 cells (1 × 106

dose-dependent manner.

**Figure 11.** The antagonistic effects of ETS vs LAPTM4B-35 overexpression on expression of oncogenes and tumor sup‐ pressor genes in HCC.

**Figure 12.** Molecular mechanism of ETS for targeting LAPTM4B-35.

#### **4. 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 Tyr285, 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.
