Oncogene

Since its discovery in a metastatic lymph node in breast cancer patients, LIM and SH3 Domain Protein (LASP1) has been shown to be over-expressed in and promote the progression of various cancers. We recently demonstrated that LASP1 is highly expressed in human Papillomavirus positive (HPV+) cervical cancers where it promotes cell proliferation and invasion. Importantly, we showed that the HPV E7 oncoprotein increased LASP1 expression by downregulating the microRNA miR-203, which directly targets the LASP1 mRNA 3UTR. However, whether LASP1 is regulated by other mechanisms in HPV+ cervical cancers is unclear. Here, we demonstrate an additional mechanism by which HPV E7 regulates LASP1 transcription. Our data demonstrates an important the role for Rb/E2F1 signalling in promoting LASP1 expression in HPV+ cervical cancer cells. Mechanistically, E7-mediated Rb binding and degradation is required for E7-driven LASP1 promoter activity. Overexpression of Rb decreased LASP1 promoter activity, LASP1 mRNA expression and LASP1 protein levels, whereas E2F1 expression promoted LASP1 expression. Importantly, E2F1 directly bound to the LASP1 promoter region and the E2F1 binding sites are essential for LASP1 expression in HPV+ cervical cancer cells. Finally, we demonstrate that LASP1 can partially rescue the growth defects observed in E2F1 knockdown cervical cancer cells. Taken together, our data show that HPV E7 regulates LASP1 expression via the Rb/E2F1 signalling pathway. In combination with our previous work, our studies demonstrate that HPV E7 employs multiple mechanisms to drive LASP1 expression, reinforcing the importance of LASP1 in HPV+ cervical cancer.

Cancer metabolism rewiring is one of the hallmarks of cancer, enabling cancer cell survival in a nutrient deprived microenvironment. Key to this is nutrient scavenging where cancer cells rely on extracellular proteins, including extracellular matrix (ECM) components, to sustain their proliferation. ECM uptake is mediated by 2{beta}1 integrin, however it is not clear how this process is controlled by nutrient availability. Here we demonstrated that amino acid starvation promoted ECM internalisation, by inducing the expression of 2 integrin. Mechanistically, starvation-driven RAS/MAPK pathway activation in cells harbouring oncogenic RAS mutations and mTOR inhibition increased 2 integrin, while the GCN2-depedent integrated stress response was not required. Functionally, elevated 2 integrin levels promoted cell adhesion and migration in nutrient starved cells. Finally, 2 integrin was found upregulated in pancreatic tumours and correlated with poor prognosis in pancreatic adenocarcinoma patients. Together, these data indicate that the nutrient- starved pancreatic cancer microenvironment synergises with KRAS mutation to drive pancreatic cancer aggressiveness.

TP53 mutations occur in 80-90% of triple-negative breast cancers (TNBCs) and drive genomic instability and metastatic progression. Poly (ADP-ribose) polymerase (PARP) is critical for DNA repair and replication fork stability. How oncogenic signaling influences PARP function to sustain proliferation during replication stress remains unclear. Mutant p53 (mtp53) R273H associates tightly with chromatin, forms complexes with PARP, and enhances PARP recruitment to replication forks [1-3]. The C-terminal region of mtp53 mediates mtp53-PARP and mtp53-Poly (ADP-ribose) (PAR) interactions that facilitate S phase progression [4, 5]. The PARP inhibitor talazoparib (TAL) combined with the alkylating agent temozolomide (TMZ) produces synergistic cytotoxicity selectively in mtp53, but not wild-type p53 (wtp53), breast cancer cells and organoids. Herein we evaluated the mechanism of mtp53-associated cell death and tested if this could translate to a preclinical xenograft model. We found that TMZ+TAL treatment induced elevated cleaved PARP and {gamma}H2AX and reduced the metastasis-promoting oncoprotein MDMX. In orthotopic xenografts expressing mtp53 R273H, but not wtp53, combination therapy significantly decreased circulating tumor cells (CTCs) and lung metastases. Transcriptomic profiling of tumors from combination treated animals demonstrated downregulation of MDMX, VEGF, and NF-{kappa}B, consistent with the observed suppression of CTCs and lung metastasis, and increased {gamma}H2AX, indicative of replication stress in mtp53 xenografts. Inhibition of metastasis was also observed in mtp53 R273H WHIM25 and p53-undetectable WHIM6 TNBC patient-derived xenografts (PDX). The mtp53 C-terminal domain (347-393) demonstrated a critical tumor promoting function, as CRISPR-mediated deletion impaired replication fork progression, tumor growth, and metastatic dissemination. DNA fiber combing showed that expression of full-length mtp53 R273H, but not C-terminal deleted {Delta}347-393, supported sustained single-stranded DNA gaps (ssGAPs) following Poly (ADP-ribose) glycohydrolase (PARG) inhibition. These findings support that mtp53 uses C-terminal amino acids to exploit PARP to enable replication stress adaptation and that mtp53 is a predictive biomarker for combined PARP inhibitor and DNA damaging therapies targeting TNBC. Significance statementTP53 mutations are the most common genetic alterations in TNBC and a major driver of replication stress and metastasis. This study shows that missense mutant p53 uses C-terminal amino acids to reprogram PARP activity to maintain tumor cell survival under replication stress. We demonstrate that p53 status governs the response to combined PARP inhibitor (PARPi) and DNA-damaging chemotherapy, establishing an additional molecular basis beyond BRCA1 mutations for treating TNBC with PARPi therapy. These findings reveal a previously unrecognized mechanism by which the mutant p53-PARP axis enables replication stress tolerance and drives cancer metastasis. We show mutation of p53 in TNBC provides an additional biomarker-guided framework to improve PARPi therapeutic outcomes.

The PBAF is one of three biochemically distinct BAF chromatin remodelers in humans. We previously proposed the role of ARID2, a PBAF component, as a bonafide tumor suppressor in colorectal cancer (CRC). Here, we validated loss of tumor suppression under conditions of ARID2 deficiency emanating from a marked reduction in PBAF complex assembly resulting from destabilization of PBAF-specific components BRD7, PHF10, and PBRM1. Transcriptome profiling of ARID2 deficient CRC cells revealed perturbation of disease processes, including CRC and neurodegenerative disorders, as well as CRC relevant pathways including Wnt/{beta}-catenin signalling, but transcript levels of PBAF-specific components remained unchanged, confirmed by RT-qPCR and TCGA data analysis. Our study establishes ARID2 as a critical stabilizer of the PBAF complex of relevance to CRC.

Cancer testis antigens are widely expressed in human malignancies. Melanoma-Associated Antigens (MAGE) A3 and A6 have been proposed to modulate protein turnover and metabolism in cancer cells. However, the substrate specificity of MAGE-A3/6 and the impact on cancer cell behavior remain poorly understood. Although previous research has identified binding partners, a molecularly validated target for MAGE-A3/6-mediated proteasomal degradation has not been described. In this study, we redefine the substrate specificity of MAGE-A3/6 and present a mechanistic framework for substrate binding, polyubiquitination, and subsequent degradation. We identify BPTF-Associated Protein of 18kDa (BAP18) as a bona fide novel substrate of MAGE-A3/6 and demonstrate its direct regulation via a molecularly defined substrate-degron-E3-adaptor interaction. The degradation of BAP18 by MAGE-A3/6 underlies phenotypic alterations in cancer cells, such as enhanced migratory capacity. This previously unrecognized molecular link is observed in both cancer cell lines and human cancer tissues, supporting a role as a fundamental oncogenic process. The discovery of a molecularly defined interaction between MAGE-A3/6 and their substrate enables systematic investigation into oncogenic protein degradation in human cancers and may inform future therapeutic strategies that leverage the molecular function of aberrantly reexpressed germline proteins in cancer.

Y-box binding protein 1 (YB-1; YBX1) is a multifunctional DNA- and RNA-binding protein involved in cell cycle regulation, DNA repair, stress adaptation, and therapy resistance. Elevated YBX1 mRNA expression is associated with aggressive disease across multiple cancers, yet its pan-cancer genomic and clinical correlates remain unclear. Here, we performed a comprehensive pan-cancer analysis across 53 datasets spanning 33 tumour types, integrating RNA expression, somatic mutations, copy number, hypoxia, and clinical outcomes. YBX1 was rarely mutated or amplified, indicating that oncogenic relevance is primarily driven by its expression. Tumours with high YBX1 mRNA exhibited a conserved transcriptional program enriched for cell cycle, DNA repair, and chromatin regulation pathways, and were preferentially mutated in genes involved in maintaining genomic stability, including TP53. These tumours were associated with increased mutation burden, fraction of genome altered, homologous recombination deficiency, and elevated hypoxia. Clinically, high YBX1 mRNA associated with advanced stage, higher grade, shorter progression-free survival, and reduced overall survival. Collectively, high YBX1 mRNA expression defines a conserved, genomically unstable, and clinically aggressive tumour state across multiple cancer types.

Despite the availability of several FDA-approved therapies, metastatic melanoma remains a significant clinical challenge, particularly for patients with brain metastases, which frequently represent the site of treatment failure and a major cause of melanoma-related mortality. Melanoma exhibits a strong propensity to metastasize to the brain, yet the molecular mechanisms driving this lethal progression remain incompletely understood, limiting the development of effective treatment options. Building on our prior discovery that focal adhesion kinase (FAK) is a key mediator of AKT1-driven brain metastasis, we sought to validate the role of FAK in melanoma progression and metastatic dissemination. Using complementary autochthonous and syngeneic mouse models of BRAF-mutant melanoma, we evaluated the impact of FAK expression on overall survival, primary tumor growth, and metastasis. Through the generation of targeted FAK mutants, we distinguished kinase-dependent from kinase-independent functions and demonstrate that FAK promotes melanoma metastasis in a kinase-dependent manner. Furthermore, we establish that FAK functions downstream of PTEN to drive metastatic progression. Collectively, these findings support the therapeutic potential of FAK inhibition, either alone or in combination with existing treatments, to more effectively combat metastatic melanoma and inform the development of emerging FAK-targeted therapies.

Ionizing radiation can be an effective therapy for prostate cancer. Unfortunately, however, more aggressive prostate cancers such as neuroendocrine prostate cancer (NEPC) are often radiation resistant, which contributes to their high degree of morbidity and mortality. In this study, we used an unbiased approach to identify novel mechanisms that contribute to resistance to radiation and that are associated with neuroendocrine differentiation. Specifically, we compared the expression of cell surface proteins by mass spectrometry in prostate cancer cell lines that had been either untreated or treated with radiation to induce resistance, a process that also promotes neuroendocrine differentiation. Among the proteins identified by this screen, we focused on folate receptor (FR) because of its known biological functions and the fact that it is a validated therapeutic target. Our data reveal that FR has a causal role in enabling prostate cancer cells to resist radiation. Importantly, we also demonstrate that the expression of FR is regulated by HIF-1, which also has a causal role in radiation resistance and neuroendocrine differentiation. Given that the ability of cells to resist damage and death in response to ionizing radiation depends largely on their ability to buffer the substantial increase in reactive oxygen species (ROS) that is generated by radiation, we also demonstrate that the folate-FR axis promotes radiation resistance by sustaining intracellular glutathione levels that buffer this increase in ROS. In summary, the data reported here highlight a novel role for FR in resistance to ionizing radiation that is intimately associated with the hypoxic microenvironment of NEPC and the ability of the folate-FRa axis to maintain redox homeostasis.

Childhood cancer is frequently associated with inherited pathogenic variants of cancer predisposition genes. Using Whole-Exome Sequencing, we identified an inherited, monoallelic pediatric cancer-linked germline variant of DNA helicase FANCJ/BRIP1, BRIP1R162Q of unclear clinical significance. Intriguingly, in vitro helicase assays demonstrated that BRIP1R162Q encodes a hyperactive DNA helicase. In cells, stable expression of BRIP1R162Q in BRIP1-proficient cells selectively sensitized to ectopic DNA replication stress. Moreover, BRIP1R162Q expressing cells showed chronic, steady-state activation of DNA replication stress indicated by decreased fork speed, fork stalling and impaired fork symmetry. Moreover, BRIP1R162Q expression triggered genomic instability indicated by elevated {gamma}H2AX foci numbers and chromosomal aberrations, suggesting BRIP1R162Q as a variant driving genomic instability. Mechanistically, BRIP1R162Q mislocalizes upon replication stress, and leads to accumulation of DNA secondary G-quadruplex (G4) structures and R-loops. RNaseH1 expression, which resolves R-loops, also reduced G4 levels and relieved BRIP1R162Q-induced replication stress. Finally, BRIP1R162Q introduces exploitable vulnerabilities for targeted therapies using G4-ligand Pyridostatin and DNA damage kinase ATR and DNA-PK inhibition. Our findings propose a mechanistic framework of how a childhood cancer-linked monoallic, hypermorphic BRIP1 germline variant participates in cancer development, and suggest potential therapeutic treatment strategies.

La-related protein 1 (LARP1) is an RNA-binding protein that post-transcriptionally regulates mRNA with potential oncogenic role in multiple cancers; however, its function in endometrial cancer remains unknown. An analysis of the TCGA endometrial cancer cohort showed that overexpression of LARP1 is associated with shorter overall survival (OS) and progression-free interval (PFI) as indicated by Kaplan-Meier analysis. Functional in vitro studies revealed that LARP1 knockdown by two different siRNAs markedly suppressed cell viability and triggered apoptosis, as confirmed by increased protein levels of cleaved PARP1 and cleaved caspase-3. Mechanistically, LARP1 knockdown remarkably reduced E2F1 protein levels as confirmed by immunofluorescence and Western blotting. Clinically, co-overexpression of LARP1 and E2F1 further decreased OS and PFI, suggesting a co-operative oncogenic axis. Importantly, LARP1 knockdown enhanced the sensitivity of ISHI and HEC-1A endometrial cancer cell lines to carboplatin treatment. These findings suggest that LARP1 promotes endometrial cancer survival and resistance to chemotherapy, at least in part, through the regulation of E2F1 and suppression of apoptosis. Targeting LARP1 could represent a promising therapeutic strategy to suppress tumor growth and enhance sensitivity to platinum-based chemotherapy.

AbstractRecurrent loss-of-function mutations in RUNX1 occur in estrogen receptor-positive (ER+) breast cancers, yet how RUNX1-loss contributes to breast tumorigenesis remains unclear. Here we used genetically engineered mouse models with luminal mammary epithelial cell (MEC)-restricted gene disruption to investigate its role in breast cancer initiation. Loss of RUNX1 alone, or together with RB1, was insufficient to drive tumor formation. In contrast, combined loss of RUNX1 and p53 induced mammary tumors with full penetrance. These tumors contained ER+ cancer cells and exhibited extensive T cell and macrophage infiltration, indicative of an immune hot microenvironment. Mechanistically, RUNX1-deficiency activated interferon signaling in luminal MECs, associated with derepression of RUNX1 target STAT1 and enhanced inflammatory responses. Consistent with these findings, human ER+ breast cancers with low RUNX1 expression displayed elevated immune signatures and poorer patient survival. Together, our results identify RUNX1-loss as a driver of an immune-active subtype of ER+ breast cancer.

The metastatic process initiates with collective cell invasion into surrounding tissues and axillary nodes, and subsequent colonization at a distant site. Previously, we found collective invasion is augmented during the G2 cell cycle phase, facilitated through Aurora kinase A (AURKA)-mediated centrosome polarization in the leader cell. Here, we identify cell cycle-associated gene signatures as overrepresented in axilla and liver metastatic sites, with AURKA expression strongly correlated with breast cancer metastasis signatures, and pan-cancer patient survival. Then, we show GFP-AURKA expression endows breast epithelia cells with the ability to form metastatic outgrowths within immune-incompetent chicken embryos. Multi-parametric imaging of wound closure assays reveals phenotypes enabled by, and dependent upon AURKA expression. We discover leader cells express AURKA and acquire front-polarized centrosomes, which differentiates them from other cells in the migrating group. Ectopic expression of GFP-AURKA induces a leader cell phenotype. Conversely, inhibition of AURKA activity alters actin dynamics, promotes turnover of cell contacts, and reduces coordination within migrating groups. Specifically, AURKA interacts with the actin regulator EPLIN, and AURKA inhibition localizes EPLIN to lamellipodia and away from E-cadherin-positive contacts. Inhibiting these necessary roles for AURKA may provide a critical barrier against the metastatic spread of human breast carcinoma cells.

Macrophages/osteoclasts are highly fusogenic cells that interact closely with bone-metastatic breast cancer cells. These cancer cells adapt to bone microenvironments by undergoing osteomimicry, acquiring bone-like phenotypes. Exploration using human breast cancer-bone metastases dataset revealed that a small population of epithelial breast cancer cells express osteoclast-like and osteomimicry genes at the single-cell level. Cell fusion and cell-in-cell (CIC) processes are two uncommon yet prognostically significant mechanisms in cancer. We showed that co-culture between murine breast cancer cells and osteoclasts yielded a unique osteoclast phenotype through dynamic cell-in-cell (CIC) interactions and fusion-like behaviours between pre-osteoclasts/mature osteoclasts and breast tumor cells, resulting in osteoclast-tumor hybrid-like cells. These tumor cell interactions characterized by membrane retention and nuclear adjacency to host nuclei were consistently observed throughout osteoclast differentiation. Single-cell sequencing analysis and interpretative assays on hybrid-like cells revealed altered extracellular matrix (ECM) modification processes, immunoregulatory, and cancer-associated pathways compared to unfused osteoclasts. Tumor cells co-cultured with osteoclasts expressed hematopoietic and osteoclast-lineage factors more strongly than tumor cells cultured alone with their effects amplified under direct cell-cell contact. The presence of these hybrid-like cells was validated in human breast cancer-bone metastases. We propose that disseminated bone-tropic breast cancer cells were stimulated by osteoclasts to undergo a non-canonical, dynamic osteoclast differentiation and CIC formation to form hybrid-like cells that may facilitate bone metastatic lesions.

The central nervous system (CNS) is a common site of metastatic spread for both non-small cell and small cell lung cancer, yet the therapeutic strategies to prevent and decrease lung cancer brain metastases remain limited. Tyrosine kinase inhibitors have shown promising results in increasing the overall response in brain metastases, owing to their brain penetrance and increased effectiveness; however, their use is limited to the small group of tumors carrying specific oncogenic drivers. Among these, inhibitors with activity against neurotrophic tyrosine receptor kinases (NTRKs) are showing promising effects in reducing CNS metastases in cancers driven by gene rearrangements of these drugs targets. However, wild-type NTRKs are susceptible to activation by their canonical ligands, which are expressed throughout the brain metastatic niche and can, in a paracrine manner, activate NTRK function in cancer cells. Here we show that NTRKs are expressed in primary tumors, brain metastases, and lung cancer cells with various driver mutations expressing wild-type NTRK2 (WT-TrkB). We demonstrate that WT-TrkB activates downstream signaling and proliferation in response to exogenous BDNF and conditioned media from reactive astrocytes known to secrete BDNF in the brain niche. Importantly, the FDA-approved NTRK inhibitor entrectinib blocked BDNF and astrocyte-induced survival pathways in multiple lung cancer cell lines, decreased their proliferation in vitro, and effectively prevented brain metastatic colonization and progression in vivo without significant effects on extracranial disease. Thus, these studies suggest that brain-dependent activation of NTRK is critical for brain metastases of WT-NTRK+ lung cancers, and therefore, NTRK inhibitors can be used to target non-fusion NTRK function to prevent or decrease brain metastases. SIGNIFICANCEThese studies demonstrate that NTRK wild-type receptors are important drivers of brain metastatic colonization and progression in different subtypes of lung cancer, independent of their driver alterations. Thus, they provide rationale to expand the use of FDA-approved NTRK inhibitors with brain penetrance for the prevention of CNS metastases.

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

Osteosarcoma is a malignant bone tumor with a high risk of metastatic relapse and poor outcomes due to primary and acquired chemoresistance. This highlights the medical need to develop effective targeted approaches to overcome chemoresistance. Recent studies have revealed the roles of metabolic reprogramming and mitochondria-nucleus crosstalk in osteosarcoma progression, indicating the potential of these cellular processes as therapeutic targets. The complex formed by mitochondrial apoptosis-inducing factor (AIF) and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) orchestrates the import and oxidative folding of cysteine-rich, nuclear-encoded proteins, thereby regulating key mitochondrial functions and metabolism. Here, we identified mitoxantrone as an inhibitor of the AIF/CHCHD4 mitochondrial import machinery and revealed a new mitoxantrone-induced metabolic vulnerability in some osteosarcoma cell line models, characterized by intracellular glutamine accumulation and an increase in nucleotide synthesis. As a result, synergy was found between mitoxantrone and the glutaminase inhibitor telaglenastat in both in vitro and in vivo osteosarcoma models. Collectively, our findings position the AIF/CHCHD4 complex as a druggable therapeutic target and provide a combination strategy for mitoxantrone/telaglenastat treatment to overcome metabolic adaptations and chemoresistance in osteosarcoma. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/716303v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1229e58org.highwire.dtl.DTLVardef@1c9af45org.highwire.dtl.DTLVardef@120d2borg.highwire.dtl.DTLVardef@11e8216_HPS_FORMAT_FIGEXP M_FIG C_FIG

Immunosuppressive tumor microenvironment (TME) inactivates CD8+ cytotoxic lymphocytes (CTLs). Here, we identify SPTBN2 spectrin as a key immunosuppressive regulator induced in CTLs in response to nutritional deficit. In human pancreatic and colorectal cancers, SPTBN2 expression negatively correlated with CTL infiltration and patients survival. In TME of mouse pancreatic and colorectal adenocarcinomas, SPTBN2 inactivated intratumoral CTLs, stimulated tumor growth and conferred cross-resistance to anti-cancer therapies. SPTBN2 knockout protected CAR T-cells from trogocytosis and increased their memory state. SPTBN2 maintained levels of cell surface proteins such as BTLA that undermine CAR T-cell cytotoxicity and promote exhaustion. Re-expression of BTLA largely reversed phenotypes in SPTBN2-deficient CAR T-cells. In manufactured CAR T cells, SPTBN2 was associated with their clinical failure in pediatric patients with leukemia. Accordingly, ablation of SPTBN2 in CAR T-cells increased their cytotoxicity, in vivo persistence and therapeutic effects indicating that SPTBN2 can be targeted to increase the efficacy of anti-cancer therapies.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy, with therapeutic resistance influenced by a dense desmoplastic stroma dominated by cancer-associated fibroblasts (CAF). Using single-cell RNA-sequencing and gene regulatory network modeling of 42 PDAC tumors, we identified a CAF subpopulation characterized by elevated NFATC2 expression that is enriched in patients with improved therapeutic response and survival. NFATC2+ CAFs exhibited tumor-suppressive features, including enhanced apoptotic signaling and suppression of ERBB pathway activity. Co-culture experiments demonstrated that NFATC2+ CAFs restrain pancreatic cancer cell growth and enhance chemotherapy-induced apoptosis, increasing sensitivity to standard-of-care chemotherapy regimens and synergizing with ERBB-targeted therapies. The favorable effect of NFATC2+ CAFs on chemotherapy response was validated in two other PDAC cohorts and in rectal cancer. Together, these findings identify NFATC2+ CAFs as a therapy-conditioned stromal state linked to improved treatment response and uncover a context-dependent vulnerability within the tumor microenvironment that may be exploited to rationally optimize combination therapies.

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

KRAS mutations underlie many human cancers. While inhibitors such as Sotorasib and Adagrasib targeting KRAS mutants have shown promise, additional strategies are required to address the broader spectrum of KRAS-driven cancers, particularly those displaying drug resistance. Thus, there is a need to better understand KRAS signaling and develop new therapeutic strategies. Here we show that KRAS4A is palmitoylated on Cys180 by a palmitoyl transferase, DHHC7 (gene name ZDHHC7). Palmitoylation promotes KRAS4A plasma membrane localization, and more importantly, nanoclustering. This in turn promotes the activation of ARAF and RAF1, but not BRAF. DHHC7 and KRAS4A Cys180 palmitoylation are important for the normal and anchorage independent growth of pancreatic cancer cell lines. Depletion of ZDHHC7 dramatically inhibits pancreatic tumor growth in mouse xenograft models. These studies provide new understandings about how palmitoylation regulates KRAS4A activity and suggest DHHC7 as a promising new target for KRAS mutant cancers.