Expression of the receptor for hyaluronic acid mediated motility (RHAMM) is associated with poor prognosis and metastasis in non-small cell lung carcinoma.

Wang D1, Narula N2, Azzopardi S2, Smith RS3, Nasar A4, Altorki NK4, Mittal V4, Somwar R3, Stiles BM4, Du YN2.
1Laboratory of Cellular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
2Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY 10065, USA.
3Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
4Department of Cardiothoracic Surgery, Weill Cornell Medicine, New York, NY 10065, USA.
The receptor for hyaluronic acid–mediated motility (RHAMM) is upregulated in various cancers, but its role in primary and metastatic non-small cell lung carcinoma (NSCLC) remains to be determined. Here, we investigate the clinical relevance of RHAMM expression in NSCLC. RHAMM protein expression correlates with histological differentiation stages and extent of the primary tumor (T stages) in 156 patients with primary NSCLC. Importantly, while focal RHAMM staining pattern is present in 57% of primary NSCLC, intense RHAMM protein expression is present in 96% of metastatic NSCLC cases. In a publicly available database, The Cancer Genome Atlas (TCGA), RHAMM mRNA expression is 12- and 10-fold higher in lung adenocarcinoma and squamous lung carcinoma than in matched normal lung tissues, respectively. RHAMM mRNA expression correlates with stages of differentiation and inferior survival in more than 400 cases of lung adenocarcinoma in the Director’s Challenge cohort. Of 4 RHAMM splice variants, RHAMMv3 (also known as RHAMMB) is the dominant variant in NSCLC. Moreover, shRNA-mediated knockdown of RHAMM reduced the migratory ability of two lung adenocarcinoma cell lines, H1975 and H3255. Taken together, RHAMM, most likely RHAMMv3 (RHAMMB), can serve as a prognostic factor for lung adenocarcinomas and a potential therapeutic target in NSCLC to inhibit tumor migration.

KEYWORDS: lung cancer; metastasis; prognosis; receptor for hyaluronic acid–mediated motility
PMID: 27220886

Lung cancer is the leading cause of cancer death worldwide, with over 200,000 new cases diagnosed in the United States alone in 2015. The two largest subtypes of lung cancer are adenocarcinoma and squamous cell carcinoma, which together account for about 80% of non-small cell carcinomas (NSCLS). The overall prognosis for patients with these cancers is poor—those diagnosed at stage IV have a 5-year survival rate of only 4%—because more than half of them are diagnosed after the cancer has metastasized. There are no current prognostic factors for determining which patients will develop metastases.

Current therapies for NSCLC including chemotherapy regimens and drugs that target molecular changes in cancer cells have considerable downsides. The cytotoxic nature of chemotherapy brings with it undesirable systemic side effects, and almost always ultimately fails to result in remission. Molecular therapies target mutations like those in epidermal growth factor receptor (EGFR) that only occur in about 20% of lung adenocarcinomas. Developing biomarkers for NSCLC that can identify which cancers are likely to metastasize would help prevent metastases by enabling tailored treatment earlier in the disease, while also providing a target for therapies that more successfully treat metastatic lung cancer.

Our lab has been involved in screening for genes that appear to be implicated in cancer development and metastases. One such gene is the receptor for hyaluronic acid-mediated motility (RHAMM), which is not expressed in normal healthy adult tissue outside of the placenta, testes, and thymus. RHAMM is, however, overexpressed in several cancers, including pancreatic, stomach, breast, bladder, and colon. In 2011, we showed that RHAMM promotes metastasis of pancreatic cancer in mice. That finding prompted us to look for broader implications for this gene in metastatic disease. Here, we aimed to determine whether RHAMM expression could act as a prognostic biomarker for NSCLC.

We evaluated a collection of 156 primary tumor specimens. Immunostaining for RHAMM expression in normal lung tissue revealed no RHAMM expression at all. Eighty-nine out of 156 primary tumors (57%) expressed RHAMM. In metastatic NSCLC that had spread to brain or bone, 22 out of 23 metastatic tumors (96%) strongly expressed RHAMM. RHAMM expression was also correlated with poor differentiation status and higher T stages. Both of these are considered poor prognostic factors for patients.

To further confirm that RHAMM is upregulated in NSCLC, we looked at mRNA levels in 57 paired lung adenocarcinomas and 49 pair lung squamous cell carcinomas from The Cancer Genome Atlas (TCGA). RHAMM mRNA expression was 12- and 10-fold higher in lung adenocarcinoma and squamous lung carcinoma than in matched normal lung tissues, respectively. Ninety-four percent of the lung adenomcarcinomas and 98% of the squamous cell carcinomas showed at least a two-fold increase in mRNA expression over the controls. RHAMM mRNA expression also correlated with poor differentiation stage. Survival data, however, indicated that higher RHAMM expression is predictive for poor outcomes in adenocarcinoma but not for lung squamous cell carcinoma. Some regulatory genes show different protein expression patterns than mRNA, but here the mRNA and protein of RHAMM data are consistent. RHAMM encodes for four different isoforms. We analyzed the TCGA data for each individual isoform and found that RHAMMv3 is the most common isoform in lung adenocarcinoma.

In a functional study, we knocked down the expression of RHAMM by short hairpin RNA (shRNA) in H1975 and H3255 cells. This resulted in downregulation of RHAMM. In addition, these cell lines reduced their migration ability after RHAMM being knocked down (Figure 1). This provides functional evidence that RHAMM is important for cell migration of lung adenocarcinoma.

Our findings have significant clinical implications. The current staging system for NSCLC allows for considerable variation in outcome between patients with the same stage cancer. There is a clinical need to identify biomarkers that more narrowly stage NSCLC. In addition, there are currently no prognostic factors for metastasis with this disease. We have shown that RHAMM could possibly be a prognostic biomarker that determines which patients will go on to develop metastatic disease. Given that normal cells don’t show RHAMM expression at all, this gene also has the potential to be a clinical target for treatment of metastatic or even primary NSCLC.

Acknowledgements:  We thank Geri Clark and Manasi M. Godbole for editorial help. This study was partially supported by DOD grant W81XWH-13-1-0331 and NIH grant 1R21CA173348- 01A1 to Nancy Du.