Developing Catchment Area Data Dashboards for Cancer Centers: A Stakeholder- engaged Approach.

Background: Data dashboards that can communicate complex and diverse catchment area data effectively can transform cancer prevention and care delivery and strengthen community engagement efforts. Engaging stakeholders in data dashboard development, by seeking their inputs and collecting feedback, has the potential to maximize user-centeredness. Objective: To describe a systematic, stakeholder-driven, and theory-based approach for developing catchment area data visualization tools for cancer centers. Methods: Cancer-relevant catchment area data were identified from national- and state-level data sources (including cancer registries, national surveys, and administrative claims databases). A prototype tool for data visualization was designed, developed, and tested based on the OPT-In [ O rganize, P lan, T est, In tegrate] framework. A working group of multi-disciplinary experts collected stakeholder feedback through formative assessment to understand data and design preferences. Thematic areas, data elements, and the composition and placement of data visuals in the prototype were identified and refined by working group members. Visualizations were rendered in Tableau © and embedded in a public-facing website. A mixed-method approach was used to assess the understandability and actionability of the tool and to collect open-ended feedback that informed action items for improvisation. Results: We developed a visualization dashboard that illustrates cancer incidence and mortality, risk factor prevalence, healthcare access, and social determinants of health for the Hollings Cancer Center catchment area. Color-coded maps, time-series plots, and graphs illustrate these catchment area data. A total of 21 participants representing key stakeholders [general audience (n=4), community advisory board members and other representatives (n=7), and researchers (n=10)] were identified. The understandability and actionability scores exceeded the minimum (80%) threshold. Stakeholders' feedback confirmed that the tool is effective in communicating cancer data and is useful for education and advocacy. Themes that emerged from qualitative data suggest that additional changes to the tool such as a warm color palette, data source transparency, and the addition of analytical features (data overlaying and area-resolution selection) would further enhance the tool. Integration of communication efforts and messages within a broader context is in progress. Discussion: A catchment area data resource developed through a systematic, stakeholder- driven, and theory-based approach can meet (and surpass) benchmarks for understandability and actionability, and lead to an overall positive response from stakeholders. Creating channels for advocacy and forming community partnerships will be the next step necessary to promote policies and programs for improving cancer outcomes in the catchment areas.

Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response.

Programmed death ligand 1, PD-L1 (CD274), facilitates immune evasion and exerts pro-survival functions in cancer cells. Here, we report a mechanism whereby internalization of PD-L1 in response to alterations of bioactive lipid/ceramide metabolism by ceramide synthase 4 (CerS4) induces sonic hedgehog (Shh) and transforming growth factor ß receptor signaling to enhance tumor metastasis in triple-negative breast cancers (TNBCs), exhibiting immunotherapy resistance. Mechanistically, data showed that internalized PD-L1 interacts with an RNA-binding protein, caprin-1, to stabilize Shh/TGFBR1/Wnt mRNAs to induce ß-catenin signaling and TNBC growth/metastasis, consistent with increased infiltration of FoxP3+ regulatory T cells and resistance to immunotherapy. While mammary tumors developed in MMTV-PyMT/CerS4-/- were highly metastatic, targeting the Shh/PD-L1 axis using sonidegib and anti-PD-L1 antibody vastly decreased tumor growth and metastasis, consistent with the inhibition of PD-L1 internalization and Shh/Wnt signaling, restoring anti-tumor immune response. These data, validated in clinical samples and databases, provide a mechanism-based therapeutic strategy to improve immunotherapy responses in metastatic TNBCs.

Targeting LINC00152 activates cAMP/Ca2+/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer.

Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER + ) breast cancer, constituting around 75% of all cases. However, the emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance by blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of the cAMP/PKA/CREB axis and increased expression of the TRPC1 Ca2+ channel. This causes cytosolic Ca2+ overload and generation of reactive oxygen species (ROS) that is, on the one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe2+ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels, in part by cAMP/CREB. These ultimately restore tamoxifen-dependent lipid peroxidation and ferroptotic cell death which are reversed upon chelating Ca2+ or overexpressing GPX4 or xCT. Overexpressing PDE4D reverses LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca2+/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of tamoxifen sensitization via restoring tamoxifen-dependent ferroptosis upon destabilizing PDE4D, increasing cAMP and Ca2+ levels, thus leading to ROS generation and lipid peroxidation. Our findings reveal LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.

Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors.

Mechanisms by which Porphyromonas gingivalis (P. gingivalis) infection enhances oral tumor growth or resistance to cell death remain elusive. Here, we determined that P. gingivalis infection mediates therapeutic resistance via inhibiting lethal mitophagy in cancer cells and tumors. Mechanistically, P. gingivalis targets the LC3B-ceramide complex by associating with LC3B via bacterial major fimbriae (FimA) protein, preventing ceramide-dependent mitophagy in response to various therapeutic agents. Moreover, ceramide-mediated mitophagy is induced by Annexin A2 (ANXA2)-ceramide association involving the E142 residue of ANXA2. Inhibition of ANXA2-ceramide-LC3B complex formation by wild-type P. gingivalis prevented ceramide-dependent mitophagy. Moreover, a FimA-deletion mutant P. gingivalis variant had no inhibitory effects on ceramide-dependent mitophagy. Further, 16S rRNA sequencing of oral tumors indicated that P. gingivalis infection altered the microbiome of the tumor macroenvironment in response to ceramide analog treatment in mice. Thus, these data provide a mechanism describing the pro-survival roles of P. gingivalis in oral tumors.

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. Methods: RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. Results: RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Conclusions: Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Transcriptome analysis of polyploid giant cancer cells and their progeny reveals a functional role for p21 in polyploidization and depolyploidization.

Polyploid giant cancer cells (PGCC) are frequently detected in tumors and are increasingly recognized for their roles in chromosomal instability and associated genome evolution that leads to cancer recurrence. We previously reported that therapy stress promotes polyploidy, and that acid ceramidase plays a role in depolyploidization. In this study, we used an RNA-seq approach to gain a better understanding of the underlying transcriptomic changes that occur as cancer cells progress through polyploidization and depolyploidization. Our results revealed gene signatures that are associated with disease-free and/or overall survival in several cancers and identified the cell cycle inhibitor CDKN1A/p21 as the major hub in PGCC and early progeny. Increased expression of p21 in PGCC was limited to the cytoplasm. We previously demonstrated that the sphingolipid enzyme acid ceramidase is dispensable for polyploidization upon therapy stress but plays a crucial role in depolyploidization. The current study demonstrates that treatment of cells with ceramide is not sufficient for p53-independent induction of p21 and that knockdown of acid ceramidase, which hydrolyzes ceramide, does not interfere with upregulation of p21. In contrast, blocking the expression of p21 with UC2288 prevented the induction of acid ceramidase and inhibited both the formation of PGCC from parental cells as well as the generation of progeny from PGCC. Taken together, our data suggest that p21 functions upstream of acid ceramidase and plays an important role in polyploidization and depolyploidization.

p17/C18-ceramide-mediated mitophagy is an endogenous neuroprotective response in preclinical and clinical brain injury.

Repeat concussions (or repetitive mild traumatic brain injury [rmTBI]) are complex pathological processes consisting of a primary insult and long-term secondary complications and are also a prerequisite for chronic traumatic encephalopathy (CTE). Recent evidence implies a significant role of autophagy-mediated dysfunctional mitochondrial clearance, mitophagy, in the cascade of secondary deleterious events resulting from TBI. C18-ceramide, a bioactive sphingolipid produced in response to cell stress and damage, and its synthesizing enzyme (CerS1) are precursors to selective stress-mediated mitophagy. A transporter, p17, mediates the trafficking of CerS1, induces C18-ceramide synthesis in the mitochondrial membrane, and acts as an elimination signal in cell survival. Whether p17-mediated mitophagy occurs in the brain and plays a causal role in mitochondrial quality control in secondary disease development after rmTBI are unknown. Using a novel repetitive less-than-mild TBI (rlmTBI) injury paradigm, ablation of mitochondrial p17/C18-ceramide trafficking in p17 knockout (KO) mice results in a loss of C18-ceramide-induced mitophagy, which contributes to susceptibility and recovery from long-term secondary complications associated with rlmTBI. Using a ceramide analog with lipid-selenium conjugate drug, LCL768 restored mitophagy and reduced long-term secondary complications, improving cognitive deficits in rlmTBI-induced p17KO mice. We obtained a significant reduction of p17 expression and a considerable decrease of CerS1 and C18-ceramide levels in cortical mitochondria of CTE human brains compared with age-matched control brains. These data demonstrated that p17/C18-ceramide trafficking is an endogenous neuroprotective mitochondrial stress response following rlmTBI, thus suggesting a novel prospective strategy to interrupt the CTE consequences of concussive TBI.

Targeting LINC00152 activates cAMP/Ca2+/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer.

Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER+) breast cancer, constituting around 75% of all cases. However, emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance via blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of cAMP/PKA/CREB axis and increased expression of TRPC1 Ca2+ channel. This causes cytosolic Ca2+ overload and generation of reactive oxygen species (ROS) that is, on one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe2+ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels. These ultimately induce lipid peroxidation and ferroptotic cell death in combination with tamoxifen. Overexpressing PDE4D rescues LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca2+/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of ferroptosis induction and tamoxifen sensitization, thereby revealing LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.