Combined inhibition of Ref-1 and STAT3 leads to synergistic tumour inhibition in multiple cancers using 3D and in vivo tumour co-culture models.

With a plethora of molecularly targeted agents under investigation in cancer, a clear need exists to understand which pathways can be targeted simultaneously with multiple agents to elicit a maximal killing effect on the tumour. Combination therapy provides the most promise in difficult to treat cancers such as pancreatic. Ref-1 is a multifunctional protein with a role in redox signalling that activates transcription factors such as NF-κB, AP-1, HIF-1α and STAT3. Formerly, we have demonstrated that dual targeting of Ref-1 (redox factor-1) and STAT3 is synergistic and decreases cell viability in pancreatic cancer cells. Data presented here extensively expands upon this work and provides further insights into the relationship of STAT3 and Ref-1 in multiple cancer types. Using targeted small molecule inhibitors, Ref-1 redox signalling was blocked along with STAT3 activation, and tumour growth evaluated in the presence and absence of the relevant tumour microenvironment. Our study utilized qPCR, cytotoxicity and in vivo analysis of tumour and cancer-associated fibroblasts (CAF) response to determine the synergy of Ref-1 and STAT3 inhibitors. Overall, pancreatic tumours grown in the presence of CAFs were sensitized to the combination of STAT3 and Ref-1 inhibition in vivo. In vitro bladder and pancreatic cancer demonstrated the most synergistic responses. By disabling both of these important pathways, this combination therapy has the capacity to hinder crosstalk between the tumour and its microenvironment, leading to improved tumour response.

Exploring transcriptional regulators Ref-1 and STAT3 as therapeutic targets in malignant peripheral nerve sheath tumours.

BACKGROUND: MPNST is a rare soft-tissue sarcoma that can arise from patients with NF1. Existing chemotherapeutic and targeted agents have been unsuccessful in MPNST treatment, and recent findings implicate STAT3 and HIF1-α in driving MPNST. The DNA-binding and transcriptional activity of both STAT3 and HIF1-α is regulated by Redox factor-1 (Ref-1) redox function. A first-generation Ref-1 inhibitor, APX3330, is being tested in cancer clinical trials and could be applied to MPNST. METHODS: We characterised Ref-1 and p-STAT3 expression in various MPNST models. Tumour growth, as well as biomarkers of apoptosis and signalling pathways, were measured by qPCR and western blot following treatment with inhibitors of Ref-1 or STAT3. RESULTS: MPNSTs from Nf1-Arfflox/floxPostnCre mice exhibit significantly increased positivity of p-STAT3 and Ref-1 expression when malignant transformation occurs. Inhibition of Ref-1 or STAT3 impairs MPNST growth in vitro and in vivo and induces apoptosis. Genes highly expressed in MPNST patients are downregulated following inhibition of Ref-1 or STAT3. Several biomarkers downstream of Ref-1 or STAT3 were also downregulated following Ref-1 or STAT3 inhibition. CONCLUSIONS: Our findings implicate a unique therapeutic approach to target important MPNST signalling nodes in sarcomas using new first-in-class small molecules for potential translation to the clinic.

Biomimetic stiffening of cell-laden hydrogels via sequential thiol-ene and hydrazone click reactions.

Hydrogels with dynamically tunable crosslinking are invaluable for directing stem cell fate and mimicking a stiffening matrix during fibrosis or tumor development. The increases in matrix stiffness during tissue development are often accompanied by the accumulation of extracellular matrices (e.g., collagen, hyaluronic acid (HA)), a phenomenon that has received little attention in the development of dynamic hydrogels. In this contribution, we present a gelatin-based cell-laden hydrogel system capable of being dynamically stiffened while accumulating HA, a key glycosaminoglycans (GAG) increasingly deposited by stromal cells during tumor progression. Central to this strategy is the synthesis of a dually-modified gelatin macromer - gelatin-norbornene-carbohydrazide (GelNB-CH), which is susceptible to both thiol-norbornene photopolymerization and hydrazone click chemistry. We demonstrate that the crosslinking density of cell-laden thiol-norbornene hydrogels can be dynamically tuned via simple incubation with aldehyde-bearing macromers (e.g., oxidized dextran (oDex) or oHA). The GelNB-CH hydrogel system is highly cytocompatible, as demonstrated by in situ encapsulation of pancreatic cancer cells (PCC) and cancer-associated fibroblasts (CAF). This unique dynamic stiffening scheme provides a platform to study tandem accumulation of HA and elevation in matrix stiffness in the pancreatic tumor microenvironment. STATEMENT OF SIGNIFICANCE: Hydrogels permitting on-demand and secondary crosslinking are ideal for mimicking a stiffening tumor microenvironment (TME). However, none of the current dynamic hydrogels account for both stiffening and accumulation of hyaluronic acid (HA), a major extracellular matrix component increasingly deposited in tumor stromal tissues, including pancreatic ductal adenocarcinoma (PDAC). The current work addresses this gap by developing a dynamic hydrogel system capable of simultaneously increasing stiffness and HA accumulation. This is achieved by a new gelatin macromer permitting sequential thiol-norbornene (for primary network crosslinking) and hydrazone click chemistry (for bioinert or biomimetic stiffening with oxidized dextran (oDex) or oHA, respectively). The results of this study provide new insights into how dynamically changing physicochemical matrix properties guide cancer cell fate processes.

Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target.

BACKGROUND: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. METHODS: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. RESULTS: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. CONCLUSION: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.