**6. Conclusion and prospective**

28 Hepatocellular Carcinoma – Basic Research

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

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

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

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.

**5.1 Adjusting the expression equilibrium between LAPTM4B-35 and LAPTM4B-24 can** 

As described in section 3, the expression equilibrium of LAPTM4B-35 and LAPTM4B-24 plays a pivotal role in maintaining the physiological status of cells. LAPTM4B-35 promotes resistance to apoptosis, deregulation of proliferation, enhancement of migration and invasion, and multi-drug resistance. On the other hand, up-regulation of LAPTM4B-24 increases sensitivity to induction of apoptosis and autophagocytosis. As we have demonstrated in our laboratory knockdown of the high expression of the endogenous cancer-promoting protein LAPTM4B-35 via RNAi, or elevating the relatively low expression of the cancer-inhibiting protein LAPTM4B-24 by transgenic therapy can significantly inhibit cancer growth and metastasis and thus may be applicable in cancer biotherapy. Therefore, we suggest knockdown of LAPTM4B-35 expression via RNAi or microRNA, and/or upregulation of LAPTM4B-24 expression may provide effective strategies for treatment of

**5.** *LAPTM4B* **gene and the encoded LAPTM4B-35 protein as a novel** 

**be a novel strategy for biotherapy of hepatocellular carcinoma** 

those from normal individuals (unpublished data).

other solid cancers.

**therapeutic targets** 

HCC and possibly other cancers.

**metastasis** 

*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

LAPTM4B: A Novel Diagnostic Biomarker and Therapeutic Target for Hepatocellular Carcinoma 31

I would also like to sincerely thank Professor Da Long Ma and his Colleges who have given me great encourage and help, and Dr. Michael A. McNutt who has provided great help in

Abdul-Ghani R., Serra V., Gyo¨ rffy B., Ju¨ rchott K., Solf A., Dietel M.et al. (2006). The PI3K

Adra CN., Zhu S., Ko JL., Guillemot JC., Cuervo AM., Kobayashi H., Horiuchi T., Lelias JM.,

Breuhahn K. & Schirmacher P. (2010). Signaling networks in human hepatocarcinogenesis- novel aspects and therapeutic options. *Prog Mol Biol Transl Sci*. Vol.97, pp.251-77. Buendia MA. (2002) Genetic alterations in hepatoblastoma and hepatocellular carcinoma: common and distinctive aspects. *Med Pediatr Oncol.* Vol.39, No.5, pp.530-5. Longerich T., Mueller MM., Breuhahn K., Schirmacher P., Benner A. & Heiss C. (2011).

Cheng XJ., Xu W., Zhang QY. & Zhou RL. (2008). Relationship between *LAPTM4B* gene

Deng LJ.,Zhang QY., Liu B. & Zhou RL. (2005). Relationship between LAPTM4B gene

Diehl JA., Cheng M., Roussel MF. & Sherr CJ. (1998). Glycogen synthase kinase-3beta

Fiorentino M., Altimari A., D'Errico A., Cukor B., Barozzi C., Loda M. & Grigioni WF. (2000).

He J., Shao GZ & Zhou RL. (2003), Effects of the novel gene, LAPTM4B, high expression in

Hogue DL., Ellison MJ., Young JD. & Cass CE. (1996). Identification of a novel membrane

Hogue DL., Kerby L. & Ling V.A. (1999). Mammalian lysosomal membrane protein confers

hepatocellular carcinoma. *Clin. Cancer Res.* Vol.6, No.10, pp.3966–3972. Fu D., Bebawy M., Kable EP. and Roufogalis BD. (2004a). Dynamic and intracellular

resistance in cancer. *Int J Cancer,* Vol.109, pp.174–181

overexpressing MRP1. *Oncogene* ,Vol.25, pp.1743–1752.

doi: 10.1002/ijc.26063. [Epub ahead of print]

*Oncology*,Vol.19, No.3, pp.527-32

*Sciences)*,Vol.37, No.3, pp.302-305

inhibitor LY294002 blocks drug export from resistant colon carcinoma cells

Rowley JD, Lim B. (1996) LAPTM5: a novel lysosomal-associated multispanning membrane protein preferentially expressed in hematopoietic cells. Genomics.

Oncogenetic tree modeling of human hepatocarcinogenesis. *Int J Cancer*. Mar 11.

polymorphism and susceptibility of colorectal and esophageal cancers. *Annals of* 

polymorphism and susceptibility of lung cancer, *J Peking University (health* 

regulates cyclin D1 proteolysis and subcellular localization. *Genes Dev,* Vol.12,

Acquired expression of p27 is a favorable prognostic indicator in patients with

trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug

hepatocellular carcinoma on cell proliferation and tumorigenesis of NIH3T3 cells, *J Peking University (health Sciences),* Vol.35, No.4, (December 2002), pp.348-352,

transporter associated with intracellular membranes by phenotypic complementation in the yeast Saccharomyces cerevisiae. *J Biol Chem*., Vol.271,

multidrug resistance upon expression in Saccharomyces cerevisiae. *J Biol Chem*,

use of the English language.

35(2):328-37.

pp.499-3511.

ISSN1671-167X

No.16, pp.9801-8

Vol.274, No.18, pp.12877-82

**8. References** 

recurrence. Conversely, up-regulation of LAPTM4B-24, an truncated isoform that lacks a 91 amino acid sequence at the N-terminus of LAPTM4B-35, promotes apoptosis and autophagocytosis, and therefore plays an antagonistic role in hepatocarcinogenesis. Overall, these data provide new insight into genes and proteins that are potentially important in the pathogenesis of liver carcinoma. This harbors great potential for future application as novel biomarkers for cancer diagnostics, pathological grading, progression monitoring and prognosis, and as novel molecular targets providing new strategies for biotherapy and chemotherapy of HCC.

Up-regulation of expression of the *LAPTM4B* gene and LAPTM4B-35 protein occur in a broad range of human solid cancers and are often associated with poor prognosis, indicating there is a common key role for this oncogentic gene and protein in cancer development.

Although *LAPTM4B* gene and the encoded LAPTM4B protein have been the subject of more than a decade of study, there are still important issues which remain to be resolved. These include (1) the underlying mechanism(s) responsible for up-regulation of the *LAPTM4B* gene and LAPTM4B-35 protein in neoplastic transformation; (2) the possibility LAPTM4B may be associated with cancer stem cells. Our previous studies indicate that LAPTM4B-35 over-expressing cancer cells possess characteristics typical of cancer stem cells, including apoptosis-resistance, unlimited renewal capacity, metastatic potential and multi-drug resistance. In addition, a study by Lee et al., (2010) suggests that LAPTM4B together with Gp49a, Sox4, and CD34 genes may be "stemness-related" genes in a primitive hematopoietic progenitor cell line EML, and this study demonstrates that these genes are preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells. Study of whether LAPTM4B-35 protein is a marker of cancer stem cells is therefore warranted; (3) as it has been shown in our study that LAPTM4B protein localizes at late endosomes, lysosomes, mitochondria, and the plasma membrane (unpublished data), it may be supposed that trafficking of LAPTM4B protein in cells would be very important for execution of its functions, thus it is worthwhile studying when and how LAPTM4B-35 and LAPTM4B-24 trafficking functions in normal cells and in cancer cells; (4) the detailed molecular mechanism for enhancing function of multi-drug resistance of p-gp (MDR1) by LAPTM4B-35 over-expression; (5) the underlying mechanism for metastasis promoted by LAPTM4B-35 over-expression; (6) the detailed molecular mechanism for signaling network activation by LAPTM4B-35 over-expression in HCC; (7) the sensitivity and specificity of LAPTM4B mRNA and LAPTM4B-35 protein as a marker for HCC diagnosis; (8) the underlying molecular mechanism for which targeted chemotherapy of small compounds depends on LAPTM4B-35 over-expression.
