Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxide

Abstract

Alternative splicing (AS) is a process that enables the generation of multiple protein isoforms with different biological properties from a single mRNA. Cancer cells often use the maneuverability conferred by AS to produce proteins that contribute to growth and survival. In our previous studies, we identified that amiloride modulates AS in cancer cells. However, the effective concentration of amiloride required to modulate AS is too high for use in cancer treatment. In this study, we used computational algorithms to screen potential amiloride derivatives for their ability to regulate AS in cancer cells. We found that 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxamide (BS008) can regulate AS of apoptotic gene transcripts, including HIPK3 , SMAC , and BCL‐X , at a lower concentration than amiloride. This splicing regulation involved various splicing factors, and it was accompanied by a change in the phosphorylation state of serine/arginine‐rich proteins (SR proteins). RNA sequencing was performed to reveal that AS of many other apoptotic gene transcripts, such as AATF , ATM , AIFM1 , NFKB1 , and API5 , was also modulated by BS008. In vivo experiments further indicated that treatment of tumor‐bearing mice with BS008 resulted in a marked decrease in tumor size. BS008 also had inhibitory effects in vitro , either alone or in a synergistic combination with the cytotoxic chemotherapeutic agents sorafenib and nilotinib. BS008 enabled sorafenib dose reduction without compromising antitumor activity. These findings suggest that BS008 may possess therapeutic potential for cancer treatment.

Biology of the mRNA Splicing Machinery and Its Dysregulation in Cancer Providing Therapeutic Opportunities

“…One of these derivatives; BS008, was found to induce cell cycle arrest and apoptosis in hepatocellular carcinoma cells, induce alternative splicing of apoptotic genes and decrease tumor growth in a hepatocellular carcinoma mouse model. Previously seen effects of amiloride on SR and hnRNP proteins were confirmed and extended in this study, together suggesting that amiloride-like compounds modulate AS in cancer cells via a diversity of effects on mRNA splice factors, regulating kinases and histone modifications [306].…”

“…While amiloride induced cell cycle arrest and apoptosis in leukemic cells [ 305 ], effective concentrations were too high to translate its use into the clinic. Therefore, an elegant study employing computational drug design identified potential molecular targets (SNRP70 and hnRNPI) of amiloride and this aided the development of amiloride derivatives with optimized docking properties onto the putative targets [ 306 ]. One of these derivatives; BS008, was found to induce cell cycle arrest and apoptosis in hepatocellular carcinoma cells, induce alternative splicing of apoptotic genes and decrease tumor growth in a hepatocellular carcinoma mouse model.…”

ESRP1 regulates alternative splicing of CARM1 to sensitize small cell lung cancer cells to chemotherapy by inhibiting TGF-β/Smad signaling

“…RNA splicing factors have emerged as a new class of oncoproteins and tumor suppressors, which lead to disease progression by regulating RNA subtypes in landmark cancer pathways [ 4 ]. Therefore, dysregulation of alternative RNA splicing is the foundation of cancer and provides a potentially rich source for new therapeutic targets [ 5 , 6 ]. Studies have reported that alternative splicing can significantly change the coding region of drug targets and play an important role in the treatment of tumor resistance [ 7 ].…”

The nexus of long noncoding RNAs, splicing factors, alternative splicing and their modulations

“…The spliceosome machinery has been directly targeted as a potential global anticancer therapy [ 139 ]. Amiloride, a drug typically used with other medications to treat high blood pressure, treatment of cells results in changes in alternative splicing [ 140 , 141 ]. Numerous oncogenic genes, including BCL-X , HIPK 3 and RON/MISTR1 , had altered alternative splicing patterns after treatment of cells with amiloride, most likely as a result of hypophosphorylation of SRSF1 [140,141].…”

“…Additionally, it has been discovered that certain small compounds can prevent the phosphorylation of particular SR proteins, and this way regulating specific alternative splicing events [ 154 ]. General splicing inhibitor, amiloride, was shown to cause hypophosphorylation of SRSF1 [ 140 , 141 ].…”