Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer.

Introduction: Triple-negative breast cancer (TNBC) comprises a heterogeneous group of clinically aggressive tumors with high risk of recurrence and metastasis. Current pharmacological treatment options remain largely limited to chemotherapy. Despite promising results, the efficacy of immunotherapy and chemo-immunotherapy in TNBC remains limited. There is strong evidence supporting the involvement of Notch signaling in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, including g-secretase inhibitors (GSIs), are quite effective in preclinical models of TNBC. However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects, since Notch plays key roles in T-cell activation, including CD8 T-cells in tumors. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and ideally, do not affect tumor immunity. We identified sulindac sulfide (SS), the active metabolite of FDA-approved NSAID sulindac, as a potential candidate to replace GSIs. Methods: We investigated the pharmacological and immunotherapeutic properties of SS in TNBC models in vitro, ex-vivo and in vivo. Results: We confirmed that SS, a known γ-secretase modulator (GSM), inhibits Notch1 cleavage in TNBC cells. SS significantly inhibited mammosphere growth in all human and murine TNBC models tested. In a transplantable mouse TNBC tumor model (C0321), SS had remarkable single-agent anti-tumor activity and eliminated Notch1 protein expression in tumors. Importantly, SS did not inhibit Notch cleavage in T- cells, and the anti-tumor effects of SS were significantly enhanced when combined with a-PD1 immunotherapy in our TNBC organoids and in vivo. Discussion: Our data support further investigation of SS for the treatment of TNBC, in conjunction with chemo- or -chemo-immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be a cost-effective, rapidly deployable therapeutic option for a patient population in need of more effective therapies.

CRISPR/Cas9 System to Knockdown MicroRNA In Vitro and In Vivo.

MicroRNAs (miRNAs) are a class of small noncoding single-stranded RNA molecules containing 18-22 nucleotides that play an important role in the regulation of gene expression at the post-transcriptional and translational levels. Loss-of-function studies are the fundamental strategy to examine miRNA function and target genes in cellular and molecular biology. Traditional methods for miRNA loss-of-function studies include miRNA-specific antisense inhibitors, miRNA sponges, and genetic knockout. However, efficiency, specificity, and stability of these methods are not adequate. Our study suggests that CRISPR/Cas9 is an economic, convenient, and innovative strategy with high efficiency, specificity, and stability for the modulation of miRNA expression. Herein, we describe a detailed protocol for knocking out miRNA genes in vitro and in vivo with the CRISPR/Cas9 system.

Genetic Editing of Long Noncoding RNA Using CRISPR/Cas9 Technology.

Long noncoding RNAs (lncRNAs) are a class of RNA transcripts greater than 200 nucleotides in length and makeup a considerable part of the human genome. LncRNAs are well established as crucial players in a myriad of physiological and pathological processes; however, despite their abundance and versatility, the functional characteristics of lncRNAs remain largely unknown predominantly due to the lack of suitable genetic editing strategies. The complexity of their genetic structure and regulation combined with their unique functionality poses several limitations in the application of classic genetic manipulation methods in lncRNA functional studies. Several reports have demonstrated the successful implementation of CRISPR/Cas9 technology in screening and identifying the function of specific lncRNAs. Here, we describe a detailed protocol utilizing CRISPR/Cas9 genetic editing technology for knocking down lncRNAs in vitro.