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. 2019 Aug 27;8:e47084. doi: 10.7554/eLife.47084

Figure 5. LDTM augments MM tumor progression.

(A) Parental KMS11 cells or cells expressing ectopic LDTM (1-470) were plated on matrigel and growth was monitored by the changes in confluence using an IncuCyte S3 instrument over 7 days. (B) KMS11 parental cells expressing DOX-inducible IRE1 shRNA or the same cells transfected with a plasmid encoding CMV-driven LDTM (1-470) were injected subcutaneously into CB-17 SCID mice. When tumors reached ~150 mm3 in volume, mice were divided into groups (n = 9) and treated with sucrose or DOX via the drinking water and tumor growth was monitored over 21 days. Tumors were harvested and lysates were analyzed by WB. (C) KMS11 parental (n = 20), LDTM overexpressing cells (n = 10), or two independent KMS11 clones harboring CRISPR/Cas9-based BAX knockout (n = 10 each) were injected subcutaneously into CB-17 SCID mice. Tumor growth was monitored over 28 days. After which, tumors were harvested and lysates were analyzed by WB. (D) Schematic model illustrating previously known (black text and lines) and novel (red text and lines) cross-regulation between the UPR and the apoptotic cascade. ER-stress-induced apoptotic signaling leads to caspase-dependent cleavage of IRE1 (1). This separates the sensing and signaling domains of IRE1, which dampens IRE1’s known XBP1s- and RIDD-mediated cytoprotective activities (2). Furthermore, it generates a fragment containing the lumenal domain and transmembrane segment (LDTM), which in turn suppresses further apoptotic signaling by attenuating BAX translocation to mitochondria (3) in a manner that can be reversed by the BCL2 inhibitor ABT-199.

Figure 5.

Figure 5—figure supplement 1. LDTM augments MM tumor progression.

Figure 5—figure supplement 1.

(A) Parental KMS11 cells or clone 13 (C13) cells expressing ectopic LDTM (1-470) were injected subcutaneously into CB-17 SCID mice (Parental n = 9, LDTM C13 n = 10) and tumor growth was monitored over 21 days. Tumors were harvested and lysates were analyzed by WB. (B) Parental KMS11 cells or a clone of LDTM (1-507) expressing cells were injected subcutaneously into CB-17 SCID mice (Parental n = 9, 1–507 C5 n = 8) and tumor growth was monitored over 21 days. Tumors were harvested and lysates were analyzed by WB.