Inhibiting NANOG Enhances Efficacy of BH3 Mimetics
The combination of gene therapy and BH3 mimetic improves the ability of either treatment alone to inhibit colony formation. Representative petri dishes are shown in which LS174T cells were cultured with either lentivrus containing shRNA to NANOGP8 (LV shNp8-1), the BH3 mimetic (ABT-737), the combination (ABT + shNp8-1), or left untreated.
BCL-2 family proteins regulate cell fate. Some members promote cell survival while others induce programmed cell death. A third group, the BH3-only members, modulates the activities of the rest of the family. Some cancers, including those of the colon and rectum, express elevated levels of pro-survival BCL-2 members, which may protect cancer cells from chemotherapy. BH3 mimetics are novel therapies that target and inhibit these pro-survival family members. Two in particular, ABT-737 and ABT-199, have activity against multiple cancer types, though neither targets the protein MCL-1, which is related to the BCL-2 family and causes resistance to the BH3 mimetics. Recent studies have revealed that the embryonic regulator NANOG and the related gene NANOGP8 can indirectly regulate MCL-1 via the kinase AKT. Abid Mattoo, Ph.D., J. Milburn Jessup, M.D., and colleagues of CCR’s Laboratory of Experimental Carcinogenesis, hypothesized that combining NANOG or NANOGP8 inhibition with a BH3 mimetic would enhance the latter’s anticancer activity.
The researchers generated lentiviruses containing small hairpin RNAs (shRNA) to inhibit NANOG or NANOGP8 expression, shNG-1 and shNp8-1, respectively. They injected mice with LS174T colorectal cancer (CRC) cells treated with either of these viruses or a virus containing a control shRNA or left untreated. Once tumors had formed, the investigators began treating the mice with ABT-737 and monitored tumor growth. Only mice receiving the combination of ABT-737 and shNG-1 or shNp8-1 had significantly reduced average tumor volumes, approximately one-third that of untreated controls.
Having supported their hypothesis, the scientists next examined the mechanism of the increased efficacy of the combination. They began by treating three CRC cell lines with ABT-737. Each cell line had a distinct sensitivity to the BH3 mimetic with CX-1 cells being most sensitive, LS174T cells being least sensitive, and Clone A cells having intermediate sensitivity. Similar results were observed after ABT-199 treatment. When the researchers analyzed the expression of pro-survival BCL-2 proteins in the cell lines, they found that LS174T cells expressed almost triple the amount of MCL-1 of the other two lines, whereas the levels of the remaining family members were similar across the three lines.
The investigators then treated the cell lines with the combinations but varied the order in which the components were given, either a low dose of ABT-737 first, shRNA-expressing lentivirus first, or both together. They observed about a 40 percent reduction in Clone A cell survival with the shNG-1 and shNp8-1 combinations with ABT-737 no matter the order. LS174T cells, however, had decreased survival only when the shNG-1 and shNp8-1 lentiviruses were given first or at the same time as ABT-737. Interestingly, the combinations only reduced survival of the CX-1 cells when the lentivirus was applied first. Because this cell line was so sensitive to ABT-737, the scientists thought the effect of NANOG or NANOGP8 inhibition might be masked. In fact, a dose response experiment with shNp8-1 lentivirus-treated CX-1 cells showed that NANOGP8 inhibition increased the cells’ sensitivity to ABT-737 by more than 50 percent. Similar experiments with ABT-199 revealed that combinations with the shNG-1 or shNp8-1 lentiviruses also enhanced survival reduction in the three cell lines. In all cases, BH3 mimetic combinations with shNp8-1 resulted in more robust inhibition of survival than those with shNG-1. The investigators also showed that cells that survive initial exposure to the combination have reduced colony regrowth efficiency, suggesting that the combination induces a persistent inhibitory effect.
Since BCL-2 family members induce cell death via apoptosis, the researchers examined the activity of executioner caspases in the treated CRC cell lines. While ABT-737 alone significantly increased Caspase-3/7 activity in CX-1 cells and to a lesser extent in Clone A cells, none of the lentiviruses activated these proteins in any of the cell lines. The combination of ABT-737 with the control shRNA lentivirus moderately increased Caspase-3/7 activity, but combinations with shNG-1 or shNp8-1 lentivirus significantly enhanced executioner caspase activity in all three lines. Blocking Caspase-3 activation with a peptide inhibitor restored cell survival in the presence of shNp8-1 lentivirus and ABT-737, demonstrating that the enhanced activity of the combination was caspase dependent.
The scientists then asked what affect the lentiviruses containing shNG-1 or shNp8-1 had on protein expression and signaling in the CRC cells. In agreement with previous studies, they found reduced levels of NANOG, phosphorylated AKT, and MCL-1 protein but not messenger RNA. To test whether the enhanced efficacy of the combination was due to the loss of MCL-1, the researchers treated CRC cells with a MCL-1 small interfering RNA (siRNA) with or without a BH3 mimetic. The MCL-1 siRNA alone increased Caspase-3/7 activity in the LS174T cells but not in the Clone A or CX-1 cells, and combining the siRNA with a BH3 mimetic significantly enhanced caspase activity in each cell line compared to the BH3 mimetic alone. In contrast, overexpressing MCL-1 in LS174T cells in the presence of shNp8-1 lentivirus and ABT-737 rescued cell survival.
Together these studies present a rationale for a new regimen targeting the pro-survival members of the BCL-2 family by inhibiting NANOG and/or its related gene NANOGP8 and using BH3 mimetics to reduce the survival and stem cell properties of CRC.Summary Posted: Wed, 10/01/2014
Mattoo AR, Zhang J, Espinoza LA, Jessup JM. Inhibition of NANOG/NANOGP8 downregulates MCL-1 in colorectal cancer cells and enhances the therapeutic efficacy of BH3 mimetics. Clin. Cancer Res. September 10, 2014 PubMed Link