Abstract
Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAFV600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAFV600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAFV600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.
Highlights
IntroductionMultiple mechanisms have been shown to contribute to v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor resistance in melanoma cells.[1,2,3,4] These include those leading to insufficient inhibition of MEK/extracellular signal-regulated kinase (ERK) signaling and those promoting melanoma cell survival and proliferation alternative to the MEK/extracellular signal-regulated kinases (ERK) pathway, such as increased activation of the PI3K/Akt or NF-kB pathway.[6,7,8,9,10,11] combinations of BRAF inhibitors and inhibitors of MEK, such as trametinib, necessary to further inhibit MEK/ERK signaling have yielded promising results in clinical trials.[12,13,14] Co-targeting the PI3K/
We report here that cotreatment with Histone deacetylase (HDAC) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors activates the caspase cascade and the mitochondrial apoptotic signaling, it kills BRAFV600E melanoma cells predominantly by induction of necrosis in a receptor-interacting protein kinase 1 (RIPK1)- and RIPK3-independent manner
The above results extend our previous finding that HDAC and BRAF inhibitors synergistically induce cell death of BRAFV600E melanoma cells by showing that, the combination triggers activation of the caspase cascade and the mitochondrial apoptotic signaling, it kills BRAFV600E melanoma cells primarily by induction of necrosis through a mechanism that is independent of RIPK1 and RIPK3
Summary
Multiple mechanisms have been shown to contribute to BRAF inhibitor resistance in melanoma cells.[1,2,3,4] These include those leading to insufficient inhibition of MEK/extracellular signal-regulated kinase (ERK) signaling and those promoting melanoma cell survival and proliferation alternative to the MEK/ERK pathway, such as increased activation of the PI3K/Akt or NF-kB pathway.[6,7,8,9,10,11] combinations of BRAF inhibitors and inhibitors of MEK, such as trametinib, necessary to further inhibit MEK/ERK signaling have yielded promising results in clinical trials.[12,13,14] Co-targeting the PI3K/. Akt and MEK/ERK pathways is being evaluated in early clinical studies.[9,15] In addition, inhibition of HSP90, a chaperon involved in regulating conformation of many kinases including mutant BRAF and Akt, has been demonstrated to overcome BRAF inhibitor resistance in melanoma cells.[16]. Our past results have suggested that sensitivity to induction of cell death may be a major determinant of long-term responses of BRAFV600E melanoma cells to BRAF inhibitors.[10] Killing of melanoma cells by BRAF or MEK inhibitors involves regulation of anti- and prosurvival proteins of the Bcl-2 family, in particular, Bim and Mcl-1.17–20 induction of melanoma cell death by inhibition of MEK has been shown to be caspase-independent, the caspase cascade is activated upon MEK inhibition in sensitive cells.[21]. Histone deacetylase (HDAC) inhibitors are emerging as a promising class of compounds in the treatment of cancer with low in vivo side-effect profiles.[22,23] monotherapy with HDAC inhibitors is not superior to dacarbazine (DTIC) in the treatment of melanoma,[24,25] combinations of HDAC inhibitors and other therapeutic agents are currently being evaluated.[26,27] Similar to cell death induced by inhibition of BRAF or MEK, induction of melanoma cell death by HDAC inhibitors involves regulation of various Bcl-2 family proteins including Bim and Mcl-1.28,29 HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) can induce caspase-independent cell death[30,31]
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