Inflammasome Sensor NLRP1 Confers Acquired Drug Resistance to Temozolomide in Human Melanoma

Abstract

Cancer cells gain drug resistance through a complex mechanism, in which nuclear factor-κB (NF-κB) and interleukin-1β (IL-1β) are critical contributors. Because NACHT, LRR and PYD domains-containing protein (NLRP) inflammasomes mediate IL-1β maturation and NF-κB activation, we investigated the role of inflammasome sensor NLRP1 in acquired drug resistance to temozolomide (TMZ) in melanoma. The sensitivity of melanoma cells to TMZ was negatively correlated with the expression levels of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme to repair TMZ-induced DNA lesions. When MGMT-low human melanoma cells (1205Lu and HS294T) were treated with TMZ for over two months, MGMT was upregulated, and cells became resistant. However, the resistance mechanism was independent of MGMT, and the cells that acquired TMZ resistance showed increased NLRP1 expression, NLRP inflammasome activation, IL-1β secretion, and NF-κB activity, which contributed to the acquired resistance to TMZ. Finally, blocking IL-1 receptor (IL-1R) signaling with IL-1R antagonist decreased TMZ-resistant 1205Lu tumor growth in vivo. Although inflammation has been associated with drug resistance in various cancers, our paper is the first to demonstrate the involvement of NLRP in the development of acquired drug resistance. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, NLRP1 might be a suitable therapeutic target in drug-resistant melanoma, as well as in other cancers.

NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma

“…To test the effects of VEM and TRA on IL-1β secretion, we used 1205Lu cells, which carry BRAF V600E mutation and have a relatively high IL1B mRNA expression, as well as IL-1β secretion among human metastatic melanoma cells ( Figure S1 ) [ 5 ]. As shown in Figure 1 a, in contrast to the effect of TMZ that significantly enhanced IL-1β secretion [ 12 ], VEM and TRA displayed an opposite effect and abrogated IL-1β secretion. Similar effects were observed in BRAF V600E -mutant A375 cells treated with TMZ, the BRAF inhibitor PLX-4720, and the MEK inhibitor CI-1040 ( Figure 1 b).…”

“…Similar effects were observed in BRAF V600E -mutant A375 cells treated with TMZ, the BRAF inhibitor PLX-4720, and the MEK inhibitor CI-1040 ( Figure 1 b). Western blot data show that a single dose of VEM and TRA suppressed ERK phosphorylation and NLRP1 protein expression, whereas TMZ increased NLRP1 protein expression [ 12 ] ( Figure 1 c and Figure S2 ). These data suggest that the MAPK/ERK pathway regulates NLRP1 protein expression and IL-1β secretion.…”

“…The elevated levels of IL-1β secreted from these SK-MEL-28 cells indicate that NLRP inflammasomes are activated in resistant cells, leading to cellular IL-1β processing and release into the extracellular space. NLRP1 and NLRP3 are two key NLRP family members for IL-1β secretion in melanoma [ 12 , 19 ]. Since increased nuclear ATF4 protein ( Figure 3 e and Figure S9 ) and ATF4 gene ( Figure 3 f) expression were seen in drug-resistant cells, we determined ATF4-regulated NLRP1 gene expression.…”

“…We then used other VEM-resistant cells derived from 1205Lu cells ( Figure 4 d and Figure S12 ) and verified the elevated PKA IIβ regulatory phosphorylation. TMZ-resistant counterparts [ 12 ] were used as control. When parental 1205Lu cells were treated with a single dose of TMZ, VEM, or TRA, we found decreased PKA IIβ regulatory phosphorylation by signaling inhibitors, VEM and TRA, but not TMZ ( Figure 4 e and Figure S2 ), consistent with the findings in Figure 1 c. However, in VEM-resistant 1205Lu cells, PKA IIβ regulatory phosphorylation was increased compared to its parental and TMZ-resistant counterparts and was further upregulated with VEM treatment ( Figure 4 f and Figure S8 ).…”

“…Therefore, we hypothesized that NLRP1 contributes positively to the development of drug resistance in human melanoma. Recently, we demonstrated the involvement of NLRP1 in the acquired resistance of metastatic melanoma cells to the chemotherapy temozolomide (TMZ) by amplifying IL-1β signaling and activating nuclear factor κB (NF-κB) activity [ 12 ]. In the present study, we further demonstrate, for the first time, that NLRP1 functions downstream of the MAPK/ERK signaling and contributes to acquired targeted therapy resistance in melanoma.…”