While melanoma represents only around 5% of all cutaneous malignancies, it accounts for the majority of skin cancer deaths due to its ability to metastasize, particularly to the lungs.

Disseminated tumor cells (DTC) from an original melanoma must proliferate and colonize the secondary site to become overt during metastasis, and the regulation of DTC growth at the secondary site determines metastatic outcome. Cell-surface proteins play an important role in this process by facilitating interactions between tumor cells and the cancer microenvironment.

In the new study, a team of researchers from the Wellcome Sanger Institute applied CRISPR activation technology to screen a library of guide RNAs targeting membrane-encoding genes to identify cell-surface molecules whose upregulation enhances metastasis of cancer cells to the lungs in vivo.

The scientists performed experimental metastasis assays in mice. They used a melanoma cell line that was virally transduced with a CRISPR guide RNA (gRNA) library targeting genes that encode membrane proteins.

Lung samples collected after 19 days had enrichment of a subset of gRNAs, suggesting positive selection for cells carrying these gRNAs during the growth of the metastases. To identify which gRNAs were significantly enriched in these mice, the team used two approaches.

The first was a percentile ranking (PR) approach, based on single gRNA abundance amongst multiple mice. The second was a joint analysis of CRISPR/Cas9 knockout screens (JACKS) analysis, which considered the relative abundance of all gRNAs for each gene in tumors collected from multiple mice.

The percentile ranking approach produced two top genes: LRRN4CL and solute carrier family 4 members 3 (SLC4A3). The JACKS analysis identified the top genes as LRRN4CL and transmembrane 4L six family member 19 (TM4SF19).

"Prior to this study, there was nothing in the scientific literature to link the LRRN4CL gene to cancer, much less to suggest that it plays such a pivotal role in metastasis," explained author David Adams, PhD, senior group leader at Wellcome. "Part of the power of CRISPR screens is that they don't require a clear hypothesis to create new insights. This is an important discovery that marks LRRN4CL as a promising drug target to help prevent the spread of cancer to the lungs and improve outcomes for patients."

LRRN4CL increases lung metastasis

The team chose to further characterize the LRRN4CL gene because it was a top hit in both analyses. LRRN4CL is predicted by UniProtKB (database containing functional information on proteins) to be a single-pass type I integral membrane protein with a signal peptide, large extracellular domain, small transmembrane/helical domain, and a short cytoplasmic tail.

The researchers expressed the LRRN4CL complementary DNA (cDNA) in three mouse melanoma cell lines and observed increase pulmonary metastatic colonization with upregulated LRRN4CL expression. When the scientists expressed the human LRRN4CL cDNA in two human melanoma cell lines, A375 and MeWo, they also observed increased pulmonary metastatic colonization relative to control cells.

Next, the team expressed LRRN4CL cDNA in three nonmelanoma mouse cancer cell lines (colorectal cancer cells, bladder cancer cells, and breast cancer cells) and again observed increased pulmonary metastatic colonization. This confirmed that LRRN4CL mediates pulmonary colonization from melanoma and other tumor types.

"Metastasis is a complex phenomenon, and metastatic cells can have different characteristics depending on the original cancer type, the secondary tumour location and even the age of the patient," said first author Louise van der Weyden, PhD, senior staff scientist at Wellcome. "However, our findings show that when metastatic cancer cells of the skin, colon or breast express high levels of LRRN4CL, it makes them uniquely able to survive and grow in the lung."

Characterizing the gene

The team determined that LRRN4CL's effect is not due to an increased ability to extravasate (flow out) from the circulation to the lung nor an ability to avoid natural killer cells, T cells, and B cells. As upregulation of LRRN4CL only provided an advantage to metastatic tumor cells in the lung, the researchers performed RNA sequencing of human A375 melanoma cells expressing LRRN4CL that had been colonizing the lung for 21 days to look for differentially expressed genes.

Principal component analysis revealed that high expression of LRRN4CL was significantly correlated to 13 downregulated genes involved in interferon (IFN) signaling pathways. Furthermore, the decreased expression of these genes is correlated with worse survival outcomes of melanoma patients.

Conversely, of the differentially expressed genes that were significantly upregulated, the team found that many have known roles in promoting metastasis, including LOXL1, MMP28, HAS1, and FPR1. The upregulation of these genes due to enhanced LRRN4CL expression promotes lung-specific metastasis.

LRRN4CL may be an attractive drug target given that it is a gene that encodes cell surface proteins; a drug only needs to be delivered to the exterior of the cell rather than the interior, making drug design less complex.

"Our results suggest that reducing the expression of the LRRN4CL gene could help to prevent metastasis to the lungs, which would already make it a potential drug target," said author Anneliese Speak, PhD, and senior scientific manager at Wellcome. "The added bonus is that this gene is expressed at very low levels elsewhere in the body, so hopefully targeting LRRN4CL wouldn't have severe side effects for patients."

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