Softening Up Chemo-Resistant Cancer Cells

DNA-damaging agents (DDAs) make up the most widely used group of cancer drugs. Yet their therapeutic success has been curtailed by drug resistance – either present in cancer cells from the disease onset or arising during treatment.

Now, scientists at the University of California San Diego (UCSD) have identified a gene that is involved in controlling the sensitivity of cancer cells to DDAs. Their investigations found that the Schlafen 11 protein impacts on the function of the protein serine/threonine kinases ATM and ATR – which play key roles in cells’ DNA damage response – by cleaving the transfer RNA (tRNA) tRNA-Leu-TAA.

“We found that if you expose cells that have Schlafen 11 to DNA damaging agents, the Schlafen 11 protein gets activated and suppresses the synthesis of ATM and ATR – that’s essentially what kills the tumor cells,” says David Schwarz, Ph.D., a professor in the biological sciences section of molecular biology and UCSD Moores Cancer Center. “In cells that do not express Schlafen 11, you do not get this downregulation of ATM/ATR and that essentially allows the tumor cells to survive.”

Understanding how cells evade the effects of DDAs will aid in the design of new strategies for resensitizing cancer cells to these drugs, suggests Jean Wang, Ph.D., professor emeritus in UCSD’s School of Medicine. “These results suggest two ways to enhance the killing of cancer cells by DNA-damaging drugs by adding ATR inhibitors or tRNA inhibitors. The paper is also of significance to the basic research on DNA damage response because it shows for the first time that regulation of tRNAs determines when a damaged cell will survive or die.”

Dr. Schwarz, Dr. Wang, Manqing Li, Ph.D., assistant project scientist at UCSD, and colleagues report their findings in Nature Structural and Molecular Biology, in a paper titled, “DNA damage-induced cell death relies on SLFN11-dependent cleavage of distinct type II tRNAs.”

SLFN11 belongs to the Schlafen (SLFN) family of mammalian genes, the first of which was discovered in mice by Dr. Schwarz back in 1998. Dr. Schwarz named the gene Schlafen, which is the German word for sleep, because its protein can stop cells from dividing. The human Schlafen gene counterpart SLFN11 was identified by Drs. Schwarz and Li four years later, and was found to encode a protein that inhibits the replication of HIV in infected human cells by blocking viral protein synthesis, but without affecting overall protein synthesis. The protein’s activity was linked with atypical codon usage in the viral RNA.

Based on this prior work in HIV, the researchers reasoned that SLFN11 may also play a role in cancer drug response. “Based on our knowledge gained from the study of SLFN11 in HIV protein synthesis, we hypothesized that SLFN11 may sensitize cells to DNA damage by inhibiting the synthesis of proteins vital to survival after DNA damage if the corresponding genes also harbor deviant codon usage,” they write. This reasoning was supported by more recent studies. “Two large-scale transcriptome profiling approaches revealed a clear requirement of SLFN11 in cancer cells for DDAs to trigger cell death,” they comment.

The team’s newly reported studies in cancer cell lines first showed that silencing SLFN11 expression rendered cells resistant to multiple DDAs. And whereas ATR and ATM expression were downregulated in cancer cells exposed to the DDA camptothecin, in cells lacking SLFN11, ATR and ATM expression weren’t affected by CPT exposure. “In contrast, both ATR and ATM messenger RNA (mRNA) levels stayed constant or were upregulated on CPT treatment,” the authors note. Interestingly, blocking ATR or ATM expression using small interfering RNAs in either cancer cells with normal, or inhibited SLFN11 expression, restored the sensitivity of SLFN11-deficient cell lines to CPT treatment, whereas silencing ATM expression had no such effect. “The inherently SLFN11-deficient pancreatic tumor cell line, MIA PaCa-2, was also sensitized to CPT treatment by siRNA-mediated suppression of ATR expression, corroborating these observations,” the authors report. Chemically blocking ATR kinase activity similarly and dose-dependently sensitized SLFN11-deficient cells to CPT treatment.

The team’s prior work demonstrating codon usage-dependent selective translational inhibition of HIV-1 had provided initial evidence that SLFN11 affects cellular tRNA levels. The latest reported tests in cancer cell lines “unexpectedly” showed that in SLFN11-expressing cells, treatment using CPT or other DDAs led to a rapid decline in levels of type II tRNAs. This group of RNAs includes all the leucine tRNAs. In contrast, there was no change in levels of type II tRNAs in SLF11-deficient cells following treatment with the DDA CPT. And expression of type I tRNAs was also unaffected in response to DDA treatment, regardless of SLFN11 expression.

Further work indicated that SLFN11-dependent downregulation of tRNA-Leu-TAA specifically inhibited protein expression of genes with a high frequency of codon T (Leu) use, such as ATR.  Although tRNA-Leu-TAA has very low abundance in cells, the authors note – “the overall TTA (Leu) codon usage frequency is only about 8% for the human genome coding sequences, and 2% for the 24 most highly expressed human cellular proteins” – the corresponding codon is used with high frequency in the ATM and ATR genes. And leading on from the finding that knocking down ATR expression sensitized SLFN11-deficient cells to DDA, the team also showed that using specially developed antisense oligonucleotides known as gapmers to directly knock down tRNA-Leu-TAA in both SLFN11-expressing, and SLFN11-deficient cells led to “profound inhibition of ATR protein expression.”

DDAs are among the most widely used anticancer treatments that account for almost one third of all chemotherapeutic drugs, the authors write. Nevertheless, many tumors are resistant to anticancer approaches based DNA-damaging treatments, and even tumors that are initially responsive will typically acquire resistance. The new studies hint at mechanisms that could be harnessed to resensitize cells to DDA. “In this article, we describe a novel molecular mechanism by which SLFN11 sensitizes cells to apoptosis upon DNA damage,” the researchers suggest. “The SLFN11-dependent downregulation of type II tRNAs, most importantly tRNA-Leu-TAA, predisposes genes that are essential for the DNA damage response and repair, such as ATR or ATM, to translational inhibition as they use the corresponding codon TTA (Leu) … Our more detailed analysis reveals that the frequency of TTA (Leu) codon usage is the apparent common denominator that subjects the encoded proteins to translational suppression by SLFN11.”

The team concluded that their findings provide new insights into the molecular mechanisms underlying SLFN11 function, but also indicate that “the direct targeting of tRNA-Leu-TAA offers a new strategy to overcome tumor cell resistance to DDAs and may hold unanticipated clinical potential.”