Mehta / Drake / Singal / Hoshida

Principal Investigators: Anand Mehta, D Phil; Richard Drake, Ph.D.
Institution: Medical University of South Carolina, Charleston, SC

Principal Investigators: Amit Singal, M.D.; Yujin Hoshida, M.D., Ph.D.
Institution: University of Texas Southwestern, Dallas, TX

Our project entitled "Aberrantly Secreted Glycoproteins as Markers of Liver Cancer" is focused on the discovery and development of biomarkers of liver cancer. This project is lead by Dr. Anand Mehta who is joined by Drs. Timothy Block and Pamela Norton of Drexel University College of Medicine, Drs. Robert Gish and Yuko Kono of the University of California, San Diego and Dr. Eiji Miyoshi of Osaka University Graduate School of Medicine.

There is a growing body of evidence suggesting that certain cancers of the GI system, such as liver, are associated with an increase in fucosylation. Surprisingly, where these fucosylated proteins arise is unknown. Recently, evidence has been presented that core fucosylated glycoproteins in hepatocytes are preferentially sorted into the bile. In contrast, non-fucosylated glycoforms of the same protein are proposed to be secreted into the circulation. The hypothesis that core fucosylation is related to sorting for apical secretion is supported by evidence from our collaborator, Dr. Miyoshi.

One explanation for the elevation of liver-derived fucosylated proteins in the circulation of HCC patients is that hepatocytes can lose their polarity and exhibit altered adhesive properties. It is further reasoned that fucosylated proteins are normally not secreted basolaterally into the sinusoids, but loss of hepatocyte polarity and/or adhesion will result in their presence in the circulation.

In our recent analysis of HCC tissue, we observed increased levels of tetra-antennary glycans versus the surrounding cirrhotic liver and normal liver. Tetra-antennary N-linked glycans arise from the action of the enzyme MGAT-5. There are reports in the literature that increased MGAT-5 expression correlates with reduced intercellular adhesion of epithelial cells. These published observations and our findings lead us to propose the following working model. We speculate that HCC is associated with an increase in tetra-antennary glycan addition to proteins involved in cell-cell adhesion and /or tight junction integrity. The resulting weaker cell-cell junctions permit “leaking” of core fucosylated glycoproteins from the biliary system, into the blood. This model would account for the apparent discrepancy between core fucose levels present in the serum (and elevated in HCC) versus tissue where no difference is observed between tumor and non-tumor. This model will be tested by over- and under-expression of the enzymes involved in glycan branching. Subsequently, this information will be used to discover novel biomarkers of HCC and validate our leads in a large cohort of clinically relevant samples obtained from our clinical collaborators from the University of California at San Diego.

Synopsis of Research and Network Resources

Brief Summary of the State of the Science and Research Needs:

Hepatocellular carcinoma (HCC) is a malignancy of hepatocytes that arises within the liver. This cancer occurs in the background of patients with underlying liver disease such as liver fibrosis and liver cirrhosis. Approximately 80% of all liver cancers are HCC and it is one of the most common malignancies worldwide. The survival rate of people with primary cancers of the liver and intra-hepatic bile ducts is very low, usually less than 2-7%. The low survival rates have been attributed to late diagnosis  and, although liver transplantation is the preferred option for surgical treatment of HCC, the paucity of organ donors means that common (worldwide) treatment is partial hepatic resection. Importantly, as a result of late diagnosis, only 5-10% are eligible for transplant or resection and thus HCC is consequently responsible for over 700,000 deaths annually (a conservative estimate) and ranks as the 2nd leading cause of cancer death worldwide.

Like most cancers, the prognosis for HCC patients is related to tumor stage at time of diagnosis, with higher rates of curative treatment and better overall survival among those with early stage tumors. Therefore, HCC surveillance has been recommended in at-risk patients using ultrasonography, with or without serum levels of the oncofetal glycoprotein, alpha-fetoprotein (AFP). However, there has been extensive debate about the utility of AFP given its suboptimal sensitivity and specificity. Thus, there has been a great desire to identify new molecules that could be used as biomarkers for HCC, particularly biomarkers with high sensitivity and specificity for early stage HCC.

Laboratory specific studies to meet the research needs:

Changes in glycosylation have been associated with the development of HCC. One such modification is increased core fucosylation on AFP. This modification is the basis of the only FDA approved test for the measurement of HCC. This assays is commercially available and is referred to as AFP-L3.  The limitation of the AFP-L3 assay is the requirement of a cancer to produce AFP protein, which only 40-60% of hepatic cancers express. To address this limitation, we have identified other proteins that contain increased levels of fucosylation and other N-linked glycan modifications. We are currently in the process of commercializing these into tests that can be used clinically as soon as possible. To aid in this development we have made several recombinant sugar binding proteins (lectins) that can be used to detect specific protein glycoforms in plate-based and fluid assays. 

In addition, although increased levels of core fucosylation have been observed in HCC for a number of years, it is unclear why these increases occur. We have been examining this and have determined that both increased levels of N-linked glycan branching and increased levels of fucosylation can be observed directly in HCC tissue. Interestingly, we have noticed that different changes in N-linked glycosylation may be related to specific genetic alterations associated with cancer development. 

Resources and Reagents for sharing:

As stated, we have developed several novel recombinant lectins and enzymes that can be used to analyze protein glycoforms in many different situations. Lectins currently available are recombinant wild type and mutant Aleuria aurantia lectins (AAL), recombinant Aspergillus oryzae l-fucose-specific lectin (AOL), recombinant Burkholderia cenocepacia lectin (BC2L), and the Psathyrella velutina mushroom lectin (PVL) to name but a few.

Public Health Implication/Advancing the Field of Glycobiology:

We are developing non invasive biomarkers for the early detection of HCC. Although HCC in the USA was once thought to be an uncommon cancer, the rates are increasing dramatically.  In 2000, there were an estimated 10,000 cases of liver cancer in the USA. Amazingly, by 2013 that number has more than tripled with over 34,000 cases annually and almost an equal number of deaths. Indeed, the occurrence of liver cancer is predicted to continue rising in the United States and if the trends continue will exceed 50,000 cases by the year 2025, leading to the same number of deaths in the USA as breast cancer.  Thus, the development of a non invasive marker that can assist in the early detection of HCC in at risk individuals will have a tremendous public health benefit.

In terms of the field of Glycobiology, we are examining the nature of the change in glycosylation observed in HCC and determining the biochemical and genetic factors associated with these changes. Our findings are applicable to other cancers where glycan changes also occur.

Cancer-Specific Relevance: detection, prevention and treatment:

Our approach to biomarker development and use involves the combination of several factors. First, our identified glycoforms are not used in isolation but in combination with AFP and other clinical factors to create biomarker algorithms that can be used to better predict cancer development.

It is important to note that current guidelines by the American Association for the Study of Liver Disease (AASLD) suggest imaging modalities for the routine surveillance of those at risk for HCC, without the use of a serum biomarker.  Unfortunately, the poor performance of ultrasound has discouraged its use and many patients are now routinely monitored by either yearly computed tomography (CT-Scan) or by Magnetic resonance imaging (MRI). However, these methods are expensive and in the case of a CT-Scan, are considered a moderate- to high-radiation diagnostic technique. As patients can live with the risk of developing HCC for 30+ years, this level of exposure is, in many ways, not acceptable. Add to the fact that these methodologies can have poor sensitivity (40-50% per lesson) for early tumors (<2.0 cm), it is clear to see why a new model is needed.

We propose the use of our novel serum glycoprotein biomarkers, which have been shown to have greater sensitivity than AFP alone, to be used with ultrasound to help identify those patients who require further examination by MRI or CT scan and improve in the early detection of cancer.

Opportunities for Collaboration:

As mentioned, we are always looking for collaboration and especially in the development of plate based assays for the detection of specific protein glycoforms.