Proximity Labeling, Quantitative Proteomics, and Biochemical Studies Revealed the Molecular Mechanism for the Inhibitory Effect of Indisulam on the Proliferation of Gastric Cancer Cells

Abstract

Indisulam exhibits antitumor activity against several cancer cells. Although the DCAF15-indisulam-RBM39 axis has been well documented in the inhibition of cancer cell growth, it is unknown whether RBM39 degradation alone is the mechanism of action of indisulam. Here, we verified the inhibitory effect of indisulam on the proliferation of gastric cancer cells and its dependence on DCAF15. Proximity-dependent biotin labeling with TurboID and quantitative proteomics revealed that indisulam indeed promoted the interaction between DCAF15 and RBM39. Immunoblotting and immunofluorescence also revealed that indisulam promoted the ubiquitin-mediated RBM39 degradation and RBM39 colocalized with DCAF15 in the nucleus. DCAF15 knockdown almost completely abolished the indisulam-mediated RBM39 reduction. Further knockdown of RBM39 eliminated the effect of DCAF15 on the proliferation of gastric cancer cells upon indisulam treatment. Immunoblotting of gastric tumor tissues confirmed the downregulation of RBM39 by indisulam. Database analysis unveiled that RBM39 was highly expressed in gastric cancer tissues and its high expression significantly shortened the survival time of gastric cancer patients. Taken together, we demonstrated that indisulam enhanced RBM39 ubiquitination and degradation by promoting its interaction with DCAF15, thus inhibiting the proliferation of gastric cancer cells. This work may provide valuable information for drug discovery through proteolysis targeting chimeras. MS data were deposited in ProteomeXchange (Dataset identifier: PXD024168).

The aryl sulfonamide indisulam inhibits gastric cancer cell migration by promoting the ubiquitination and degradation of the transcription factor ZEB1

“…Consistent with previous discoveries ( 32 ), RNA-binding motif protein 39 (RBM39) is the most significantly downregulated protein. It has been reported previously that RBM39 mediates the indisulam-induced cytotoxicity in several types of cancer cells ( 27 ) and the indisulam-inhibited proliferation of gastric cancer cells ( 32 ). Therefore, we focused on other potential targets.…”

“…To explore the potential biological processes that indisulam might regulate in gastric cancer cells, we used label-free quantitative (LFQ) proteomics to identify proteins that are differentially regulated by indisulam following the procedure described in Figure 1 A . Briefly, AGS cells expressing DCAF15 from a previous work ( 32 ) were treated with dimethyl sulfoxide (DMSO) (control) or indisulam and lysed. Proteins in cell lysates were reduced, blocked, digested, desalted, and analyzed by Orbitrap Fusion Lumos Tribrid mass spectrometer.…”

“…Therefore, drugs that suppress these processes may have potentials for the therapeutic treatment of gastric cancer. Our previous studies have discovered that indisulam can significantly inhibit the proliferation of gastric cancer cells by promoting the ubiquitination and degradation of RBM39 ( 32 ). Among the identified proteins, N-cadherin is a marker for EMT and migration of cancer cells ( 33 ).…”

A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II

“…The samples for AP-MS were prepared following pulldown procedure as previously described with minor modifications 54 . Briefly, U2OS cells were lysed in RIPA buffer and the cell lysate was equally divided into two groups: (1) DMSO group (control group): the cell lysate was incubated with 20 μM biotin-labeled compound 1 ; (2) Cpmd 1 group (sample group): the cell lysate was incubated with 20 μM biotin-labeled compound 1 and 200 μM free compound 1 .…”

Research progress of proteomics in screening tumor markers of gastric cancer

“…Present the specific clusters (44992, 44993) of differential expression had potential implications for evaluating the sensitivity of radiotherapy and chemotherapy and guiding individualized drug use. Jiaqi Lu [27] revealed the molecular mechanisms by which indoquinone inhibits the proliferation of GC cells AGS, HGC27, MGC803, and SGC7901 through neighborhood labeling techniques, quantitative proteomics, and biochemical studies. Their group found that indisulam enhanced the ubiquitination and degradation of RBM39 by promoting its interaction with DCAF15, thereby inhibiting the proliferation of gastric cancer cells.…”