Loss of LGR5 through Therapy-induced Downregulation or Gene Ablation Is Associated with Resistance and Enhanced MET-STAT3 Signaling in Colorectal Cancer Cells.

Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5- state in response to chemotherapy, LGR5- targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody-drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.

An antibody-drug conjugate targeting GPR56 demonstrates efficacy in preclinical models of colorectal cancer.

BACKGROUND: Long-term prognosis remains poor for colorectal cancer (CRC) patients with advanced disease due to treatment resistance. The identification of novel targets is essential for the development of new therapeutic approaches. GPR56, an adhesion GPCR, is highly expressed in CRC tumours and correlates with poor survival. Here, we describe the generation and preclinical evaluation of a novel ADC consisting of an anti-GPR56 antibody (10C7) conjugated with the DNA-damaging payload duocarmycin. METHODS: RNA-seq dataset analysis was performed to determine GPR56 expression in CRC subtypes. The specificity of binding, epitope mapping, and internalisation of 10C7 was examined. 10C7 was conjugated to payload and ADC cytotoxicity was assessed against a panel of CRC cell lines and tumour organoids. Antitumour efficacy was evaluated in xenograft models of CRC cell lines and patient-derived tumours. RESULTS: High GPR56 was shown to be associated with the microsatellite stable (MSS) subtype that accounts for 80-85% of CRC. GPR56 ADC selectively induced cytotoxicity in CRC cells and tumour organoids at low nanomolar potency in a GPR56-dependent manner and showed significant antitumour efficacy against GPR56-expressing xenograft models. CONCLUSIONS: This study provides the rationale for the future development of a GPR56-targeted ADC approach to potentially treat a large fraction of MSS CRC patients.

Drug Conjugates of Antagonistic R-Spondin 4 Mutant for Simultaneous Targeting of Leucine-Rich Repeat-Containing G Protein-Coupled Receptors 4/5/6 for Cancer Treatment.

LGR4-6 (leucine-rich repeat-containing G-protein-coupled receptors 4, 5, and 6) are three related receptors with an upregulated expression in gastrointestinal cancers to various extents, and LGR5 is enriched in cancer stem cells. Antibody-drug conjugates (ADCs) targeting LGR5 showed a robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cancer cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4-6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 furin domain mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. Drug conjugates of a peptibody comprising the RSPO4 mutant and IgG1-Fc showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects.

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion.

GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.

The Hippo Pathway in Cardiac Regeneration and Homeostasis: New Perspectives for Cell-Free Therapy in the Injured Heart.

Intractable cardiovascular diseases are leading causes of mortality around the world. Adult mammalian hearts have poor regenerative capacity and are not capable of self-repair after injury. Recent studies of cell-free therapeutics such as those designed to stimulate endogenous cardiac regeneration have uncovered new feasible therapeutic avenues for cardiac repair. The Hippo pathway, a fundamental pathway with pivotal roles in cell proliferation, survival and differentiation, has tremendous potential for therapeutic manipulation in cardiac regeneration. In this review, we summarize the most recent studies that have revealed the function of the Hippo pathway in heart regeneration and homeostasis. In particular, we discuss the molecular mechanisms of how the Hippo pathway maintains cardiac homeostasis by directing cardiomyocyte chromatin remodeling and regulating the cell-cell communication between cardiomyocytes and non-cardiomyocytes in the heart.

RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model.

BACKGROUND: The androgen receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR-targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin-1 (IL-1), represses AR messenger RNA (mRNA) levels and activity in AR-positive (AR+ ) PCa cell lines concomitant with the upregulation of prosurvival biomolecules. Thus, we contend that IL-1 can select for AR-independent, treatment-resistant PCa cells. METHODS: To begin to explore how IL-1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL-1-treated LNCaP PCa cells. Our pathway analysis predicted nuclear factor kappa B (NF-κB) p65 subunit (RELA), a canonical IL-1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used small interfering RNA (siRNA) to silence the NF-κB family of transcription factor subunits, RELA, RELB, c-REL, NFKB1, or NFKB2, in IL-1-treated LNCaP, C4-2, and C4-2B PCa cell lines. C4-2 and C4-2B cell lines are castration-resistant LNCaP sublines and represent progression toward metastatic PCa disease, and we have previously shown that IL-1 represses AR mRNA levels in C4-2 and C4-2B cells. RESULTS: siRNA revealed that RELA alone is sufficient to mediate IL-1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL-1-mediated repression of AR than C4-2 and C4-2B cells, RELA siRNA led to a more striking derepression of AR mRNA levels and AR activity in C4-2 and C4-2B cells than in LNCaP cells. CONCLUSIONS: These data indicate that there are RELA-independent mechanisms that regulate IL-1-mediated AR repression in LNCaP cells and suggest that the switch to RELA-dependent IL-1 repression of AR in C4-2 and C4-2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.

Identification of an IL-1-induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR- PCa cells.

BACKGROUND: In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL-1 may also contribute to AR-targeted therapy resistance. Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL-1 reprograms AR positive (AR+ ) PCa cells into AR negative (AR- ) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival and tumor progression in the inflammatory tumor microenvironment. METHODS: LNCaP and PC3 PCa cells were treated with IL-1ß or HS-5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT-QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA-PCa-2b, PC3, and DU145 PCa cell lines were treated with the IL-1 family members, IL-1α or IL-1ß, or exposed to HS-5 BMSC in the presence or absence of Interleukin-1 Receptor Antagonist (IL-1RA). Treated cells were analyzed by western blot and/or RT-QPCR. RESULTS: Comparative analysis of sequencing data from the AR+ LNCaP PCa cell line versus the AR- PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment. CONCLUSIONS: Our data supports that IL-1 reprograms AR+ PCa cells to mimic AR- PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival.