In this synthetic lethal screening system, we used oncogenic RAS (HRASG12V, although all hits were tested for oncogenic- KRAS-selectivity); we searched for compounds with increased lethality in the presence of this mutation. It is possible that some hits from such a screen might have selectivity toward HRAS, but not other RAS isoforms; such small molecules are potentially useful research tools to define differences in downstream signaling pathways activated by different RAS isoforms. Even in this case, it is important to note that the target protein for the compound would be very unlikely to be HRASG12V itself. According to the concept of synthetic lethality, one mutation is HRASG12V, and the hit compound simulates a second mutation in gene B by binding to protein B ().
This implies that a synthetic lethal screening strategy enables us to target the oncogenic-RAS-signaling pathway, without inhibiting RAS proteins themselves, which provides a broader range of targets in a single screen compared to conventional target-based in vitro screening. For example, these novel compounds (RSL5 and RSL3) display RAS-RAF-MEK-signal-dependent activity. As all three RAS isoforms can activate downstream RAF-MEK signaling cascades, the synthetic lethality of RSL5 and RSL3 is not limited to HRASG12V. We demonstrated this isoform-independent action of RSL3 by using shRNAs targeting the three RAS genes in a lung-carcinoma-derived cell line (). It should be noted that due to the remoteness of the target protein from RAS, other oncogenes may also sensitize cells to these RSL compounds if they activate signaling pathways largely overlapping with RAS.
Erastin and RSL5 exploit VDAC3 to induce synthetic lethality with oncogenic RAS. VDACs (VDAC1, VDAC2, and VDAC3) are mitochondrial pores that transport ions and small metabolites, including NADH, citrate, and succinate (Lemasters and Holmuhamedov, 2006
). The use of VDACs as cancer-cell-selective drug targets has not been addressed, except in our previous report of erastin-induced, nonapoptotic, oxidative cell death. The presence of a compound, RSL5, that uses VDACs to induce selective lethality in oncogenic-RAS-containing cancer cells emphasizes the importance of VDACs as novel targets with a sufficient degree of promiscuity in their interaction with small-molecule ligands to enable multiple scaffolds to target these proteins.
One interesting shared property of RSLs is their iron-dependent mechanism of action. Many cancer cells are reported to have an enriched iron pool (Shterman et al., 1991
); cancer patients have higher levels of iron than normal individuals (Stevens et al., 1988
). Our BJ cell system captured this relationship between iron and cancer as BJ-TERT/LT/ST/RASV12
cells have more iron than their isogenic, nontumorigenic counterparts (). The greater iron content in BJ-TERT/LT/ST/RASV12
cells, together with the iron-dependent action of RSLs, lead us to the idea that the increased iron pool within cancer cells might be a target for inducing cancer-cell-specific lethality. Indeed, investigators have explored this possibility by testing iron chelators for anticancer properties (Kalinowski and Richardson, 2005
). A number of iron chelators are reported to have good potency in inhibiting cancer cell growth. For example, bleomycin, a key component of standard chemotherapeutic regimens for treating patient with germ-cell tumors (Kondagunta and Motzer, 2006
), is known to oxidatively damage DNA through its complex with iron (Dorr, 1992
). Another iron chelator, Triapine, entered phase II clinical trials in a combination therapy with cisplatin to treat ovarian cancer (Low and Schoenfeldt, 2005
The increased iron concentration in BJ-TERT/LT/ST/RASV12
cells compared to their isogenic counterparts raised the possibility that oncogenic-RAS signaling alters iron metabolism to augment the cellular labile iron pool (). Oncogene-induced increases in-cellular iron have been reported for c-myc and E1A(Tsuji et al., 1995
;Wu et al., 1999
). Oncogenic signals from c-myc or E1A downregulate expression of the heavy subunit of ferritin (FTH1), which is a subunit of iron storage complex. Since ferritin functions as an iron buffer, downregulation of ferritin by oncogenic signals may result in an increased labile intracellular iron pool. Consequently, proliferation-driving enzymes, such as ribonucleotide reductase, are replenished with sufficient iron to function.
Oncogenic RAS has been proposed to enrich the cellular iron pool mainly by upregulation of TfR1
expression. The existence of a mitogen-responsive element in the 5′ untranslated region (UTR) of TfR1
mRNA has been reported, indicating that RAS-RAF-MEK-MAPK signaling may upregulate TfR1
(Casey et al., 1988
; Ouyang et al., 1993
). Our data are consistent with this hypothesis; BJ-TERT/LT/ST/RASV12
cells have increased mRNA and protein levels of TfR1
compared to BJ-TERT and BJ-TERT/LT/ST cells (). In addition to this TfR1-mediated pathway, we found that oncogenic RAS downregulates FTH1
to decrease the capacity of ferritin iron storage (). As c-myc and E1A were reported to downregulate FTH1
expression, it would be interesting to test whether ferritin downregulation is a common means to enrich the cellular iron pool by oncogenes. Moreover, efforts to identify target proteins of RSL3 may ultimately reveal novel proteins in iron metabolism regulation; some such proteins are likely to be connected to RAS signaling.
These data demonstrate that synthetic lethal screening makes it possible to identify small molecules with enhanced lethality toward oncogene-harboring cancer cells, without targeting the oncogene itself. This feature of synthetic lethal screening becomes useful when we seek drug leads targeting loss of function cancer mutations, such as RAS, or tumor suppressors. Mechanism of action studies with these small molecules may lead to discovery of novel pharmacological targets for treating cancer and elucidation of critical connections between the targets and the cancer genes of interest; this should allow for a refined diagram of signaling networks in tumor cells.