Inadequate nutrient intake leads to oxidative stress disrupting homeostasis, activating signaling, and altering metabolism. Oxidative stress serves as a hallmark in developing prostate lesions, and an aggressive cancer phenotype activating mechanisms allowing cancer cells to adapt and survive. It is unclear how adaptation and survival are facilitated; however, literature across several organisms demonstrates that a reversible cellular growth arrest and the transcription factor, nuclear factor-kappaB (NF-κB), contribute to cancer cell survival and therapeutic resistance under oxidative stress. We examined adaptability and survival to oxidative stress following nutrient deprivation in three prostate cancer models displaying varying degrees of tumorigenicity. We observed that reducing serum (starved) induced reactive oxygen species which provided an early oxidative stress environment and allowed cells to confer adaptability to increased oxidative stress (H 2 O 2 ). Measurement of cell viability demonstrated a low death profile in stressed cells (starved + H 2 O 2 ), while cell proliferation was stagnant. Quantitative measurement of apoptosis showed no significant cell death in stressed cells suggesting an adaptive mechanism to tolerate oxidative stress. Stressed cells also presented a quiescent phenotype, correlating with NF-κB nuclear translocation, suggesting a mechanism of tolerance. Our data suggests that nutrient deprivation primes prostate cancer cells for adaptability to oxidative stress and/or a general survival mechanism to anti-tumorigenic agents.

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which alternative signaling entities can be created; yet, how receptor heterodimers affect receptor pharmacology remains unknown. Previous observations suggested a biochemical antagonism between GPCRs, CXCR4 and CB2 (CNR2), where agonist-bound CXCR4 and agonist-bound CB2 formed a physiologically nonfunctional heterodimer on the membrane of cancer cells, inhibiting their metastatic potential However, the reduced signaling entities responsible for the observed functional outputs remain elusive. This study now delineates the signaling mechanism whereby heterodimeric association between CXCR4 and CB2, induced by simultaneous agonist treatment, results in decreased CXCR4-mediated cell migration, invasion, and adhesion through inhibition of the Gα13/RhoA signaling axis. Activation of CXCR4 by its cognate ligand, CXCL12, stimulates Gα13 (GNA13), and subsequently, the small GTPase RhoA, which is required for directional cell migration and the metastatic potential of cancer cells. These studies in prostate cancer cells demonstrate decreased protein expression levels of Gα13 and RhoA upon simultaneous CXCR4/CB2 agonist stimulation. Furthermore, the agonist-induced heterodimer abrogated RhoA-mediated cytoskeletal rearrangement resulting in the attenuation of cell migration and invasion of an endothelial cell barrier. Finally, a reduction was observed in the expression of integrin α5 (ITGA5) upon heterodimerization, supported by decreased cell adhesion to extracellular matrices Taken together, the data identify a novel pharmacologic mechanism for the modulation of tumor cell migration and invasion in the context of metastatic disease. This study investigates a signaling mechanism by which GPCR heterodimerization inhibits cancer cell migration. .

No abstract

563 Background: Muscle-invasive bladder cancer (MIBC) accounts for ~25–30% of all bladder cancer diagnoses. With neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) as standard-of-care, the 5-year survival rate ranges from 40%–60%. Bladder-sparing protocols (BSP) have emerged as a feasible alternative to RC for MIBC treatment, however better tools are needed. In this study, we evaluated the prognostic value of circulating tumor DNA (ctDNA) in predicting recurrence in patients who achieved pathological complete response (pCR). Methods: We analyzed a previously described cohort of 68 patients (656 plasma samples; Christensen et al., JCO 2019) with MIBC who received NAC prior to cystectomy. Patients had an updated median follow-up of 58.94 months (range: 7.19-81.77) post-cystectomy. ctDNA was analyzed at baseline (before NAC; N=64), and prior to cystectomy (N=65) using a commercially available assay (Signatera, Natera, Inc.). Additionally, exploratory RNA-Seq was performed on tumors from 59 patients (samples with >5M total counts were utilized). Pathway analysis was used to compare ctDNA-positive and ctDNA-negative patients who failed to achieve pCR. Results: Of the 64 patients with ctDNA results available at baseline, 59.4% (38/64) tested ctDNA-negative, and of these 84.2% (32/38) achieved pCR. Furthermore, 40.6% (26/64) tested ctDNA-positive, and only 34.6% (9/26) achieved pCR. Likewise, prior to cystectomy, 83.9% (52/62) of patients were ctDNA-negative, and 80.7% (42/52) achieved pCR, while none of the ctDNA-positive patients achieved pCR (positive predictive value 100%; negative predictive value 80.8%). Based on both ctDNA timepoints, the probability of ctDNA-negative patients to achieve pCR was significantly higher than ctDNA-positive patients ( p<0.0001). Notably, ctDNA-positive patients without pCR demonstrated significantly poorer RFS and OS compared to the ctDNA-negative patients, at both timepoints (Baseline: RFS; HR=8.2, p=0.017, OS; HR=8.4, p=0.015, prior to cystectomy: RFS; HR=5.2, p=0.0078, OS; HR=4.8, p=0.012). Furthermore, ctDNA status at baseline and before cystectomy was a better predictor of RFS compared to pCR (HR=8.5, p<0.0001, HR=14, p<0.0001, respectively). Transcriptomic pathway analysis of patients who did not achieve pCR showed an enrichment of oncogenic pathways, namely EMT and angiogenesis, in tumors from ctDNA positive patients, whereas tumors from ctDNA negative patients showed an enrichment of anti-tumor immune signatures, including IFNα and IFNγ. Conclusions: Absence of ctDNA was significantly associated with pCR both at baseline and prior to cystectomy, identifying patients who may benefit from BSP. Larger cohorts are warranted to test the prognostic value of ctDNA combined with transcriptomic profiling in informing patient selection for avoiding cystectomy.