Scientists discovered several years ago that interleukin-12, a protein that immune cells secrete in response to an invading pathogen, can also alert disease-fighting T cells to recognize, attack, and remember tumor cells for months to come. But as researchers have developed Il-12 as a possible treatment for cancer, they have found the protein or its gene is most effective when injected directly into tumors, not infused into the bloodstream.
Although researchers don’t know why this is so, some wonder whether biology might be trying to teach them a valuable lesson. They say it may be that the much higher concentration of Il-12 when applied directly to tumors prompts cells to express specific genes at higher levels and/or transmit signals along different anti-growth or suicide-inducing pathways. It also suggests that identifying these specific genes and pathways might allow scientists to amplify their tumor-shrinking effects with Il-12 or other compounds.
Now, in a key first step in this direction, a team of scientists has identified 14 genes in oral squamous cell carcinoma cells that respond in particular to direct administration of IL-12. Of these genes, the scientists say they found four in particular whose expression levels changed tremendously in response to the increased Il-12 levels. While two of the genes fall into the category of "the usual suspects," the others - IRF7 and Wsb2 - are little studied by cancer researchers and could provide excellent targets for further investigation.
"What’s nice about these data is they were generated from actual tumors in mice, not from cultured tumor cells, which often alter their normal pattern of gene expression under laboratory conditions and sometimes provide misleading clues," said Dr. Shulin Li, the lead author on the paper and a scientist at Louisiana State University in Baton Rouge. The work, published in the March 2004 issue of the journal Molecular Therapy, was supported jointly by NIH’s National Institute of Dental and Craniofacial Research and the National Cancer Institute.
According to Li, the latest paper builds on his group’s success in transferring the Il-12 gene directly into squamous cell carcinoma cells with an increasingly popular gene-transfer technique called electroporation. Unlike gene-bearing viruses that first must find a way to enter cells, not always a sure thing, scientists rely on sharp pulses of electricity that polarize lipids in the cell membrane, opening up small channels into the cell.
The researchers then inject circular bits of gene-containing DNA, called plasmids, through the holes and into the cell’s cytoplasm. Shortly thereafter, the channels close again; but the plasmids remain inside the cell, where their transfer gene is expressed, typically at high levels.
So well has the technique worked in preliminary studies, Li et al. previously reported fully eradicating four out of 10 oral squamous cell carcinomas in mice. Moreover, the scientists found the mice maintained an immune response against the tumor cells for at least 11 months following the initial gene transfer. "We view it as a one-two punch," said Li, noting that the technique can be applied in a way that causes tumor cells to malfunction and commit suicide. "That is, the electroporation itself and the plasmid vector can induce heavy tumor cell death. Then, on top of that, the Il-12 gene works to alert the immune system to the appearance of the tumor cells, allowing them to detect them as foreign."
In the latest paper, Li et al. took up the issue of direct Il-12 treatment and its greater efficacy. The scientists hypothesized that they would see changes in gene expression between two groups of mice with oral squamous cell carcinomas. The first group would have their tumors treated directly with IL-12 electroportation gene therapy; the other would receive direct electroporation but without receiving a functional gene, essentially serving as a control group.
To monitor gene expression in the two groups, they turned to an increasingly common tool in biology called a cDNA microarray. Like taking a genetic snapshot, cDNA microarrays allow scientists to record at one frozen moment in time the levels of thousands of individual genes at once, an impossibility just over a decade ago.
Li said he and his colleagues decided to place on the array 1,176 predetermined mouse genes. All were known from previous studies to be expressed in mouse tumors. However, their precise expression levels remained undetermined.
After recording the expression levels under the different treatment conditions, the group then sifted through the data in search of genes that had a three-fold or more difference in their expression levels. "With a two-fold difference, you might get, say, 100 genes but maybe half of them are false positives, meaning your lab wastes time and valuable resources," said Li. "We just felt a three-fold cutoff would be more meaningful."
Of the 14 genes that met this more stringent criteria, four had a 10-fold or more difference in their expression levels. One of them - STAT1 - is a well-known target of so-called cytokines, immune signaling proteins, such as Il-12. Scientists say STAT-1 as a molecular control switch that, when activated, activates numerous signaling pathways and prompts the transcription of multiple genes.
Nor was there surprise about the increased expression of the gene MIG, which encodes a protein that stimulates immune T cells. Previous studies have indicated that MIG seems to contribute to the antitumor effects of Il-12. "STAT1 and MIG are exactly the types of genes that one would expect to respond to the increased production of Il-12," said Li. "So, that was a good sign that the microarray were meaningful."
Li said the other two genes - Wsb2 and IRF7 - were a bit more interesting. He said cancer researchers previously have given only a cursory look at these genes, although IRF7 in particular has been much studied by immunologists for its role in regulating interferons, proteins that cells release when they are invaded by viruses.
"This suggests that there is more to the IRF-7 story than viruses," said Li. "In fact, we’ve found in our follow-up work that, if we overexpress IRF-7 in squamous cell carcinoma cells, the primary oral cancer will grow very slowly in mice, but we still need to figure out what exactly its protein product does."
Li said he and his colleagues are actively pursuing these new leads. "With tumors that are physically accessible, such as oral cancers, a real positive with electroportation itself is you can inject directly into tumors, and it takes a heavy toll on the tumor cells," said Li. "I think electroporation could have an important future role in treating oral cancer, particularly when combined with immunogene therapy, chemotherapy and/or radiation treatment, and my group will continue to explore this possibility."