**4.** *LAPTM4B* **gene and the encoded LAPTM4B-35 protein are predicted to be novel markers for diagnosis of hepatocellular carcinoma**

#### **4.1 LAPTM4B-35 is predicted to be a novel marker for diagnosis, pathological grading and progression-monitoring of hepatocellular carcinoma**

We have demonstrated via Western blot and immuno-histochemistry that the expression levels of LAPTM4B-35 in HCC tissues show a significant positive correlation with pathological grade (Figure 17a, and Table 1 and 3), intrahepatic and extrahepatic metastasis (figure 17b), TNM stage (Table 1 and 3) and recurrence of HCC (Table 3), and show a negative correlation with overall and disease-free postoperative survival (Figure 17c). Upon multivariate analysis, elevated expression of LAPTM4B-35 was found to be an independent


Table 3. Expressive level of LAPTM4B-35 in HCC tissues shown by IHC shows significant positive correlation to portal vein invasion, TNM staging and recurrence. (H.Yang et al., 2010b).

pcDNA-BE or infection with replication-deficient adenovirus Ad-BE induces apoptosis and autophagocytosis of HCC cells, as well as associated cellular and molecular alterations. At the same time LAPTM4B-24 up-regulated HCC cells lose its carcinogenicity (unpublished data). These studies indicate that LAPTM4B-24 plays an antagonistic role in

In summary, the LAPTM4B-35 plays pivotal roles in keeping cell survival, proliferation, migration and invasion, and so on; whereas LAPTM4B-24 plays critical roles in regulating programmed cell death, including apoptosis and autophagocytosis. The expressive equilibrium of LAPTM4B-35 and LAPTM4B-24 maintains physiological homeostasis of cells. Destroy of this equilibrium would lead to diseases. Up-regulation of LAPTM4B-35 leads to oncogenesis, while up-regulation of LAPTM4B-24 may plays a role in cancer regression.

**4.** *LAPTM4B* **gene and the encoded LAPTM4B-35 protein are predicted to be** 

**4.1 LAPTM4B-35 is predicted to be a novel marker for diagnosis, pathological grading** 

We have demonstrated via Western blot and immuno-histochemistry that the expression levels of LAPTM4B-35 in HCC tissues show a significant positive correlation with pathological grade (Figure 17a, and Table 1 and 3), intrahepatic and extrahepatic metastasis (figure 17b), TNM stage (Table 1 and 3) and recurrence of HCC (Table 3), and show a negative correlation with overall and disease-free postoperative survival (Figure 17c). Upon multivariate analysis, elevated expression of LAPTM4B-35 was found to be an independent

Table 3. Expressive level of LAPTM4B-35 in HCC tissues shown by IHC shows significant positive correlation to portal vein invasion, TNM staging and recurrence. (H.Yang et al., 2010b).

**novel markers for diagnosis of hepatocellular carcinoma** 

**and progression-monitoring of hepatocellular carcinoma** 

hepatocarcinogenesis.

Fig. 17. Correlation of LAPTM4B-35 expression levels in HCC tissues with pathological grading, metastasis and postoperative survival time. (a) Level of LAPTM4B-35 protein in HCCs shows positive correlation with pathological grade. (b) LAPTM4B-35 expressions in HCCs from 65 patients were divided into three groups: ''Low,'' ''Mediate,'' and ''High.'' Level of LAPTM4B-35 in HCC shows significant positive correlation with intrahepatic and extrahepatic metastasis. \*P<0.05. (c) Levels of LAPTM4B-35 in HCCs show significantly negative correlation with overall (left) and disease-free (right) postoperative survival of cancer patients. (H.Yang et al., 2010a, 2010b).

LAPTM4B: A Novel Diagnostic Biomarker and Therapeutic Target for Hepatocellular Carcinoma 29

Neoadjuvant chemotherapy generally targets one or more molecules in signaling pathways. As discussed in section 3, over-expression of LAPTM4B-35 promotes hepatocarcinogenesis, faster growth of human HCC xenografts and metastasis in mice, and leads to anti-apoptosis, deregulation of proliferation, and enhancement of migration and invasion and multi-drug resistance. In addition, over-expression of LAPTM4B-35 leads to accumulation of a number of cell cycle promoting proteins and survival proteins, and results in down-regulation of a number of cell cycle inhibiting proteins and proapoptotic proteins by activating a signaling network including at least 4 different signaling pathways. Based on our studies in vitro and in vivo, it is clear that LAPTM4B-35 has significant potential as an important novel target for cancer treatment. Recently, we have screened out a few small molecules from various libraries containing nearly 2000 chemically synthetic compounds. These small molecules are able to kill a variety of cancer cells that over-express the *LAPTM4B* gene and LAPTM4B-35 protein in vitro, but do not harm the normal fetal liver cells which do not over-express this gene. One of these small molecules has been demonstrated to inhibit growth and metastasis of human HCC xenografts in nude mice, and results in cellular and molecular alterations opposite to those that result from over-expression of LAPTM4B-35 in HCC cells. Some of these active small molecules have been demonstrated to be inhibitors of tyrosine kinase

In summary, this gene and the proteins it encodes harbor great potential for application in

*LAPTM4B* gene and its encoding LAPTM4B-35 protein are over-expressed in 87% of HCC and some solid cancers with varying frequencies. The level of LAPTM4B-35 expression in HCC tissues shows significant positive correlation with pathological grade, intrahepatic and extrahepatic metastasis, and recurrence, and negative correlation with overall and diseasefree postoperative survival of cancer patients, and is thus an independent prognostic factor for HCC. Over-expression of LAPTM4B-35 promotes malignant transformation of cell lines from normal tissues, including human liver tissue. This result implies that over-expression of the *LAPTM4B* gene and LAPTM4B-35 protein may play pivotal roles in hepatocarcinogenesis and progresion. Up-regulated LAPTM4B-35 promotes faster growth and metastasis of human HCC xenografts in nude mice, and results in resistance to apoptosis, deregulation of proliferation, and 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 encoded by oncogenes and down-regulation of a number of tumor suppressing proteins. In contrast, knockdown of endogenous LAPTM4B-35 via RNA interfering leads to significant inhibition of growth and metastasis of human HCC xenografts in nude mice, and reversion of the cellular and molecular malignant phenotypes. The extensive effects caused by LAPTM4B-35 over-expression result from its function in activation of a signaling network, including at least 4 signaling pathways that are commonly known to be associated with hepatocarcinogenesis (K. Breuhahn, 2010; Takigawa, 2008; XH , 2011). Taken together, our studies suggests that LAPTM4B-35 is a key molecule which functions upstream of a cancer associated signaling network and plays pivotal roles in hepatocarcinogenesis, progression, metastasis, multi-drug resistance and

**5.2 LAPTM4B-35 is a novel therapeutic target for neoadjuvant chemotherapy** 

(unpublished data).

cancer biotherapy and chemotherapy.

**6. Conclusion and prospective** 

prognostic factor for hepatocellular carcinoma (Yang et al., 2010a, 2010b). In our preliminary study we found that about 40% of paired non-cancerous liver tissues in the same HCC patients express elevated LAPTM4B mRNA (JJ. Liu et al., 2000). We believe that the noncancerous liver tissue of the same one patient with HCC is representing the precancer status to variant extents. We therefore suggest that LAPTM4B-35 may be an important and novel biomarker for early diagnosis, mornitoring of progression, prediction of metastasis and recurrence of HCC, as well as for evaluating prognosis of HCC patients. In addition, we have demonstrated that LAPTM4B-35 protein can be released into the blood in the form of exosomes. The levels of LAPTM4B-35 in serum of HCC patients are significantly higher than those from normal individuals (unpublished data).

Moreover, a number of other cancers also show the similar association of LAPTM4B-35 with clinical and pathological criteria. Cancers which have been evaluated include gallbladder cancer (L. Zhou et al., 2007), cholangiocarcinoma (L. Zhou et al., 2008), ovarian cancer (Y Yang, 2008; Yin et al., 2011a, 2011b), cervical cancer (Meng et al., 2010), and endometrial cancer (Meng et al., 2010). It is likely that LAPTM4B-35 may also find use as a biomarker for other solid cancers.

#### **4.2** *LAPTM4B* **mRNA expressed in circulating tumor cells can be used for predicting metastasis**

It is well known that circulating cancer cells have more highly metastatic potential. We found that the highly metastatic cancer cell lines derived from one cancer mass express higher levels of LAPTM4N-35 than syngenic cancer cell lines of low metastatic potential (XR. Liu et al., 2003). We have determined that the LAPTM4B mRNA expressed by a few circulating HCC cells can be measured by nested RT-PCR and real time RT-PCR. Therefore, testing LAPTM4B mRNA in circulating HCC cells with real time RT- PCR shows promise for application of prediction and evaluation of metastasis occurring at an early stage.
