Molecular Cancer Research

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all renal cancer cases. Despite its generally indolent behavior and low mutational burden, there is no targeted therapy for metastatic ChRCC. Profilin-1 (Pfn1), a cytoskeletal regulator of actin and tubulin dynamics, has emerged as a potential oncogenic driver in several cancers including RCC, but its role in ChRCC, remains undefined. We observed elevated Pfn1 expression in stage IV ChRCC patients, implicating Pfn1 in advanced disease progression. To investigate this, we manipulated Pfn1 expressions in two ChRCC cell lines UOK276 and RCJ41M. Pfn1 knockdown (KD) significantly reduced proliferation, invasion, and colony formation, whereas Pfn1 overexpression (OE) in UOK276 enhanced ChRCC aggressive phenotypes. Pharmacological inhibition of Pfn1 significantly suppressed proliferation and clonogenic growth in both cell lines. Additionally, Pfn1 KD increased intracellular ROS accumulation, while overexpressed reduced ROS levels, linking cytoskeletal regulation to oxidative stress control. Together, these findings position Pfn1 as a critical mediator of ChRCC progression, linking cytoskeletal remodeling to aggressive tumor behavior. This work highlights Pfn1 as a potential therapeutic target and establishes a framework for cytoskeletal-focused strategies in advanced ChRCC.

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.

The formation of the premetastatic niche prepares distant tissues for tumor cell engraftment. Endothelial cells are critical mediators of premetastatic niche formation, orchestrating extravasation of circulating tumor cells and critical pro-tumor immune cells, such as neutrophils. In mouse models of breast cancer, we show that primary tumors upregulate the non-signaling chemokine receptor ACKR1 in the endothelium of the lung premetastatic niche. ACKR1-expressing venules were found to be preferential sites of neutrophil and tumor cell localization within lung tissue. A newly generated conditional ACKR1 allele was used to show that endothelial-specific removal of ACKR1 expression significantly reduces metastatic engraftment in the lung. When ACKR1 is activated by tumor-secreted factors, endothelial ACKR1 functions to promote neutrophil recruitment within the lung parenchyma. We conclude that ACKR1 is a critical component of the endothelial response to tumors at the metastatic site of the lung, leading to neutrophil recruitment and promotion of tumor cell metastasis. SUMMARYEndothelial cells play critical roles in breast cancer metastasis. ACKR1 is upregulated in the endothelium of the lung metastatic niche in response to primary mammary tumors. Endothelial ACKR1 expression was found to promote neutrophil infiltration into the metastatic niche and support breast tumor cell metastasis to the lung.

Increased matrix metalloproteinase (MMP) expression has long been recognized as a common feature of colorectal cancers (CRCs), yet less is known about how these enzymes interact to impact cancer progression. Taking advantage of single-cell and spatial transcriptomic data, we analyzed the cell-type-specific and spatial expression of MMPs in CRCs. Distinct colon cancer-associated fibroblast (CAF) subtypes were found to express different MMP combinations, including MMP1/3-expressing and MMP11-expressing CAFs. Conversely, myeloid cells (monocytes, macrophages, and dendritic cells) expressed varying levels of the "myeloid MMPs" 9, 12, and 14, which correlated closely with secretory gene expression. Finally, a small population of cancer cells expressed high levels of MMP7. The MMP7-expressing cancer cells frequently co-expressed MMP1, MMP14, and several Wnt-related genes, consistent with a cancer cell type at high risk of malignancy and metastasis. Spatial transcriptomic data showed MMP expression in discernible clusters driven in part by cell-type localization, including fibroblast-heavy stromal regions and inflammatory cell hubs. Epithelial-rich areas showed subregions of MMP7-expressing cancer cells, including areas where cancer cell and myeloid MMP expression overlap. Tumors showed a wide variation in MMP1-expressing CAFs, a variation reflected in primary CAF cell lines. In vitro, MMP1 expression was a stable phenotype that persisted through multiple rounds of division. MMP1-expressing CAFs were frequently positioned at the stromal interface, suggesting a role in facilitating cell movement across the tumor boundary. Our analysis indicates that cell-type and positional MMP expression varies between tumors and may play a role in determining lesion progression and cancer spread.

We previously reported that the murine lncRNA Epr is essential for maintaining colon mucosal integrity and permeability. Mice lacking Epr in the colon are more susceptible to colitis and tumor development. Additionally, we demonstrated that human EPR expression is reduced in ulcerative colitis and in a small cohort of colon adenocarcinoma patients. Here, we present evidence that human and mouse EPR share several key physiological features: preferential binding to the KH1 domain of their interacting protein, KSRP; specific expression in canonical and immature goblet cells of the large intestine; and a functional role in intestinal goblet cell development. The correlation between EPR levels and survival in large cohorts of metastatic colon adenocarcinoma patients, together with the capacity of human EPR to inhibit cell proliferation and induce apoptosis in two distinct human colon adenocarcinoma cell lines, suggests that EPR may serve as both a valuable prognostic marker for goblet cell-derived adenocarcinomas and a potential therapeutic target.

Suppressor of cytokine signaling 1 (SOCS1) negative regulates inflammatory cytokine production and attenuates oncogenic growth factor signaling pathways. Reduced SOCS1 protein expression in human prostate cancer correlates with greater disease severity. To define the physiological functions of SOCS1 functions in the prostate, we conditionally ablated Socs1 in prostate epithelial cells of C57BL/6 mice. These Socs1{Delta}PE mice exhibited normal prostate development, maturation and lobular architecture. However, adult Socs1{Delta}PEmice developed progressive epithelial hyperplasia and inflammatory cell infiltration that were temporally and spatially distinct. SOCS1-deficient prostate showed increased epithelial cell proliferation and elevated oxidative stress markers, and prostate organoids recapitulated this hyperplasia phenotype. Diet-induced obesity exacerbated both hyperplasia and inflammation in SOCS1-deficient prostate. Upon transurethral infection with uropathogenic Escherichia coli UPEC1677 expressing the genotoxin colibactin, Socs1{Delta}PE mice developed invasive prostate cancer with complete loss of lobular architecture, whereas control mice developed hyperplasia and pre-neoplastic lesions. In vitro, SOCS1-deficient prostate organoid-derived epithelial cells exhibited increased DNA damage following exposure to UPEC1677. Deletion of the colibactin biosynthetic gene clbP in UPEC1677 abolished its ability to induce DNA damage in SOCS1-deficient cells and to drive prostate cancer in vivo. Proteomic analysis of prostate organoids revealed dysregulation of basal and luminal epithelial lineage markers and signaling pathway proteins that could promote neoplasia in SOCS1-deficient cells. Collectively, these findings establish an essential, epithelial cell-intrinsic role for SOCS1 in maintaining prostate tissue homeostasis by restraining proliferation, regulating lineage plasticity, limiting inflammation and oxidative stress, and conferring protection against genotoxic injury and neoplastic transformation.

Well- and dedifferentiated liposarcomas (WDLPS and DDLPS) are characterized by extensive copy- number alterations rather than recurrent gene-inactivating mutations, obscuring the molecular mechanisms that drive disease progression and, critically, the transition from well-differentiated to the more aggressive dedifferentiated tumor states. Despite marked differences in clinical behavior and prognosis, the regulatory events underlying adipocytic lineage destabilization in DDLPS remain poorly understood. Here, we establish an in vivo model of retroperitoneal liposarcoma in Xenopus tropicalis through early embryonic mosaic perturbation of p53 and Rb pathway components. Combined disruption reproducibly induced retroperitoneal WDLPS development, demonstrating that pathway-level deregulation of the MDM2-p53 and CDK4-Rb axes is sufficient to initiate liposarcoma development in vivo. Strikingly, additional perturbation of transcriptional co-activator ep300 in this context resulted in increased tumor dedifferentiation, yielding lesions composed of spatially coexisting well- and dedifferentiated adipocytic states. In contrast, direct targeted disruption of downstream adipogenic regulators recurrently lost in human DDLPS, including cebpa, g0s2, and dgat2, failed to induce dedifferentiation in the same genetic context in vivo. These findings indicate that dedifferentiation cannot be explained by loss of downstream adipocytic effectors alone but instead reflects destabilization of higher-order regulatory programs governing adipocytic identity. Together, these results establish an in vivo model that closely reflects the clinical situation on a pathway level and provides initial mechanistic insight into how adipocytic differentiation may become destabilized during disease progression. This framework offers a foundation for future studies leveraging higher-order and multi-omic approaches to dissect the molecular processes underlying the WDLPS-to-DDLPS transition.

Osteosarcoma (OS) genomes are characterized by complex genomic rearrangements (CGRs) that drive genomic instability and clonal diversification early in tumor evolution. As a result, OS tumors display high inter-patient variability, which has hindered molecular stratification and targeted therapeutic development. To study genomic complexity in OS and credential a genetically engineered mouse model of the disease (Sp7-Cre Trp53fl Rb1fl), we performed high-depth and multi-region whole genome sequencing (WGS) of 35 tumor samples from 24 mice. Similar to human OS, the murine OS tumors (mOS) had a high number of somatic structural variants (158 per tumor) with low tumor mutational burden of single nucleotide variants (0.87 mutations/MB). CGRs were identified in 63% (15/24) of mOS cases, most frequently affecting chromosome 15 (33%, 8/24 mice) and resulting in Myc amplification in 6 mice, ranging from 5 to 104 copies. Myc amplification was verified with DNA FISH, long-read sequencing and gene expression data, which revealed examples of Myc amplification in both extrachromosomal circular DNA (ecDNA) and in derivative chromosomes generated by CGRs. PTEN loss occurred frequently (59% 12/22 mice), and contributed to osteosarcomagenesis, as demonstrated by tumor initiation with in vivo CRISPR/Cas9-mediated deletion experiments (2 mice). Together, these results demonstrate that a preclinical model of osteosarcoma can generate the genomic heterogeneity and complexity of the human disease, thereby facilitating research into mechanisms of tumor initiation and drivers of progression and relapse.

Platinum resistance remains a major barrier in Ovarian cancer (OC) treatment[1]. While hyperactivation of DNA damage response (DDR) is a hallmark of chemoresistance[2], the underlying epigenetic mechanisms driving this adaptation remain poorly understood. Here, we identify a novel post-transcriptional regulatory axis involving miR-221-5p that governs two critical DDR effectors: RAD18, which mediates DNA damage tolerance through trans-lesion synthesis (TLS)[3][4], and RAD51, the central recombinase for homologous recombination (HR)[5][6]. Although the miR-221/222 cluster is traditionally categorized as oncogenic[7][8], we demonstrate that the miR-221-5p arm functions as a potent tumor suppressor in OC. Bioinformatic and luciferase reporter assays confirmed that miR-221-5p directly targets the 3'UTRs of both RAD18 and RAD51. In OC clinical specimens and cell lines, miR-221-5p downregulation inversely correlates with RAD18/RAD51 expression. Functionally, miR-221-5p restoration suppressed platinum-induced PCNA mono-ubiquitination and HR, inducing a "functional BRCAness" that sensitized both established and patient-derived primary OC cells to carboplatin and PARP inhibition. Furthermore, in vivo disseminated xenograft models demonstrated that stable miR-221-5p expression significantly reduced tumor burden. Collectively, our results delineate a novel regulatory mechanism where loss of miR-221-5p drives chemoresistance by derepressing the RAD18/RAD51 axis, identifying this axis as a promising therapeutic target.

BackgroundFibrosis and tumor innervation are two features of the tumor microenvironment (TME) that contribute directly to the lethality of pancreatic ductal adenocarcinoma (PDAC), but their potential interactions have not been explored. Moreover, although it is known that activated Schwann cells (SCs) stimulate cancer cell invasion, it remains unclear how SCs are activated. ObjectiveWe determined how SCs are activated in the pancreatic fibrotic microenvironment. DesignThe correlation between physical features of the microenvironment and SC activation was assessed in human patient samples and in mice by SC c-Jun phosphorylation monitoring, atomic force microscopy and multiphoton live imaging. Several in vitro models in which forces were applied to SCs expressing a reporter for c-Jun phosphorylation and RNA-Seq analysis were used to decipher the cellular and molecular mechanisms of SC activation. ResultsNerves surrounded by stiff stroma present higher SC activation. Intravital imaging shows a matrix dependent SC activation. Mechanical forces on SCs induce c-Jun phosphorylation in SCs in a non-canonical manner that involves a nuclear sensing machinery with the proinflammatory enzyme Phospholipase A2. ConclusionFibrosis enhances the protumorigenic impact of innervation by activating SCs via a mechanism in which nuclear compression triggers non-canonical activation of the AP-1 transcription factor complex. Pancreatic fibrosis alone, without cancer cells, is sufficient to activate SCs, suggesting this mechanism may be common across non-malignant pancreatic diseases. Notably, SCs are more sensitive to mechanical activation than PDAC cells. These findings reveal TME interactions that may guide future microenvironment-targeted PDAC therapies. What is already known on this topicThe pancreatic cancer tumor microenvironment is highly innervated and fibrotic, two components of the tumor microenvironment that regulate tumorigenesis. How they impact each other is unknown. Schwann cells have emerged as a significant protumorigenic player, but the triggers of Schwann cell activation remain undefined. What this study addsWe establish that fibrosis induces Schwann cell activation and characterize the mechanism by which it occurs. We uncovered a mechanical mode of action that deforms nuclear membrane and activates c-Jun in Schwann cells, which contradicts the traditional view of c-Jun activation through a stimulus detected at the plasma membrane. How this study might affect research, practice or policyThis study provides a better understanding of the biology of pancreatic ductal adenocarcinoma and supports the development of novel precision therapies that target the fibrotic microenvironment to impact the protumorigenic effect of tumor innervation.

The RNA polymerase-associated factor 1 complex (PAF1C) is an evolutionarily conserved transcription elongation complex that regulates RNA polymerase II-mediated transcription and chromatin modification. LEO1, a core subunit of PAF1C, has been implicated in developmental gene regulation, WNT signaling, and leukemogenesis; however, its role in solid tumor progression remains poorly understood. In this study, we found that although LEO1 expression is generally elevated in colorectal cancer (CRC), its expression is reduced in stage IV tumors and is associated with poor clinical outcomes. To investigate its function, we established LEO1 -deficient HCT116 cell line and performed transcriptomic analyses. Loss of LEO1 suppressed epithelial differentiation and developmental gene programs while inducing cell cycle delay. Despite these changes, LEO1-deficient cells exhibited aggressive phenotypes, including enlarged nuclei and increased expression of migration-associated genes, which were further enhanced under glucose deprivation. Motif analysis identified FOXM1 as a key regulator of these migration-related genes. Mechanistically, LEO1 deficiency promoted accelerated transcriptional activation of GRP78, a central regulator of endoplasmic reticulum (ER) stress adaptation. GRP78 was required for survival under ER stress conditions, and its inhibition suppressed both migration and migration-associated gene expression. In addition, transcriptomic analyses revealed upregulation of cholesterol metabolism-related genes in LEO1-deficient cells. Consistently, treatment with the HMG-CoA reductase inhibitor atorvastatin selectively impaired their survival, indicating cholesterol metabolic dependency. Collectively, these findings demonstrate that LEO1 loss promotes ER stress-adapted migration and cholesterol metabolic dependency in CRC, suggesting that these pathways may represent therapeutic vulnerabilities in metastatic LEO1-low CRC.

The standard-of-care for patients with higher-risk non-muscle invasive bladder cancer (NMIBC) after tumour resection is intravesical administration of Bacillus Calmette-Guerin (BCG). While this form of adjuvant immunotherapy has improved recurrence-free and progression-free survival, a large proportion of patients experience recurrences within a year of diagnosis. The reasons for this high rate of early recurrence following BCG therapy remain unclear; however, inadequate activation of systemic immunity may be a contributing factor. To address this, we analysed the transcriptomic and chromatin accessibility profiles of peripheral blood mononuclear cells obtained from patients with NMIBC at single-cell resolution before BCG immunotherapy and after five induction doses of BCG. Monocytes from patients who experienced disease recurrence within a year of initiation of BCG therapy (BCG non-responders) exhibited a pro-inflammatory phenotype consistent with age-related immunosenescence prior to BCG immunotherapy. Moreover, inflammation-associated pathways that were active before initiation of BCG therapy in the BCG non-responders were down-regulated after five instillations of BCG. In contrast, these pathways were quiescent before BCG therapy in patients who remained disease-free for at least a year but were markedly up-regulated after five doses of BCG. Genomic regions with accessible chromatin were enriched in activator protein 1 (AP-1) binding sequences in monocytes from BCG-non-responders prior to BCG therapy. AP-1 is a central regulator of the inflammatory phenotype associated with immunosenescence. Our findings indicate that a pre-existing state of innate immunosenescence underlies early disease recurrence following BCG. Patients unlikely to benefit from BCG may be offered alternative therapies early in their disease journey. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723215v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f7c844org.highwire.dtl.DTLVardef@7cea65org.highwire.dtl.DTLVardef@1008d23org.highwire.dtl.DTLVardef@131f973_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Keratin-positive giant cell-rich tumor (KPGCT) is a newly described bone and soft tissue tumor. The tumor is characterized by scattered keratin-positive cells and the presence of HMGA2::NCOR2 fusions. It is not known if the HMGA2::NCOR2 fusion is located in the keratin-positive cells, and little is known about how KPGCT develops. KPGCT shares some histologic features with tenosynovial giant cell tumor (TGCT), a soft tissue tumor with CSF1 rearrangements. Single-nuclei RNA sequencing (snRNA-seq) and Xenium spatial transcriptomics were used to elucidate the mechanisms driving KPGCT and compare KPGCT to TGCT. We show that the neoplastic cells in KPGCT constitute only a minority of cells in the tumor, and that they co-express keratin, HMGA2 and CSF1. The neoplastic cells in KPGCT express no synovial markers, confirming KPGCT as a distinct entity, separate from TGCT. The bulk of the tumor consists of CSF1R-expressing macrophages and osteoclast-like giant cells, suggesting an important role for CSF1-CSF1R signaling. In addition, we find that the cells with the HMGA2 translocation show activation of the hippo signaling pathway, which is known to regulate CSF1 expression. We show that the CSF1-CSF1R axis, possibly regulated through the hippo signaling pathway, plays an important role in KPGCT. This axis likely stimulates the migration and proliferation of macrophages, which form the majority of cells in the tumor, as well as their differentiation into osteoclasts-like giant cells. These results provide a rationale for the use of CSF1R inhibitors, which have already shown efficacy in TGCT, as a therapy for KPGCT. SignificanceKeratin-positive giant cell-rich tumor (KPGCT) is a rare, newly described soft tissue and bone tumor. By examining this tumour on a single-cell level, we confirm the identity of the neoplastic cells on a molecular level, showing these form a minority of cells in the tumor. We show that activation of the hippo pathway in the neoplastic cells is a likely driver of tumorigenesis. Additionally, we show the neoplastic cells produce large amounts of CSF1, attracting the macrophages that form the majority of cells in the tumor. This finding gives supporting evidence for anecdotal reports of response to CSF1 inhibitor therapy. Finally, we identify key differences between KPGCT and tenosynovial giant cell tumor, a tumor that shares histological features with KPGCT.

Clear cell renal cell carcinoma (ccRCC) exhibits heterogeneity in immune infiltration and clinical outcomes, but the mechanisms governing recruitment and organization of tumor-reactive CD8 T cells remain incompletely defined. We investigated the role of the CXCL13-CXCR5 axis in shaping CD8 T cell recruitment, differentiation, and immune organization in high-risk, non-metastatic ccRCC. Human tumor, plasma, and matched adjacent kidney specimens were analyzed using ELISA, quantitative PCR, migration assays, multiplex immunofluorescence, single-cell RNA sequencing, spatial transcriptomics, and a syngeneic mouse model. CXCL13 was among the most upregulated chemokines in ccRCC relative to matched normal kidney and was embedded within a CD8 T cell-associated inflammatory transcriptional program. In transwell and microphysiological system (MPS) assays, CXCL13 promoted CD8 T cell migration, enriched CXCR5 cells among migrating CD8 T cells and showed reduced migration after CXCL13 or CXCR5 blockade. Single-cell analyses identified CXCR5 expression within stem-like CD8 T cell states associated with TCF7 and IL7R, whereas CXCL13 associated with later cytotoxic/exhausted states along a continuous differentiation landscape. Spatial transcriptomics demonstrated that stem-like CD8 T cells localized within structured lymphoid aggregates enriched for B cells, coordinated CXCL13/CXCR5 expression, and signaling programs. In vivo, tumor-derived CXCL13 suppressed tumor growth, increased intratumoral CD8 T cell infiltration, and enriched CXCR5TCF1CD8 stem-like T cells. In human tumors, higher CXCL13 expression correlated with increased CXCR5CD8 T cell infiltration and improved recurrence-free survival. These findings identify CXCL13 as a regulator of immune recruitment and niche organization and support the CXCL13-CXCR5 axis as a biomarker and possible therapeutic target in ccRCC.

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.

During breast cancer metastasis, tumor cells migrate toward intratumoral blood vessels and intravasate through stable structures known as TMEM (Tumor Microenvironment of Metastasis) doorways. TMEM doorways, composed of a Mena-expressing tumor cell, a Tie2hi/VEGFhi macrophage, and an endothelial cell, are clinically validated prognostic markers of distant metastasis in breast cancer and represent the exclusive sites of tumor cell intravasation. We previously demonstrated that Tie2 signaling is essential for TMEM doorway function and tumor cell intravasation. In this study, we investigated how Tie2 signaling promotes tumor cell intravasation and metastasis. Because all three TMEM doorway-associated cell types can express Tie2, we sought to determine which of these cells contribute to the Tie2 signaling-dependent vascular opening at TMEM doorways and tumor cell dissemination. We found that endothelial cells associated with TMEM doorways secrete Ang2, which stimulates VEGF-A expression in Tie2hi macrophages. Elevated VEGF-A levels at TMEM doorways increase vascular permeability, facilitating tumor cell entry into the bloodstream. Using tissue staining and line-scan analysis of Tie2 and lineage markers in human and mouse breast cancer models, we observed Tie2 expression in macrophages, tumor cells, and endothelial cells. To assess functional contributions, we selectively disrupted Tie2 in macrophages, endothelial cells, and cancer cells using CRISPR-Cas9 and RNAi approaches and tested in which of these cell-knockouts of Tie2 expression affected transendothelial migration in vitro. Macrophage-specific Tie2 deletion had the greatest impact on tumor cell intravasation. To confirm this finding in vivo, we generated a mouse model with inducible, macrophage-specific Tie2 knockout. Acute, targeted loss of Tie2 specifically in macrophages significantly reduced TMEM doorway associated vascular opening and tumor cell intravasation. Together, these findings establish macrophage Tie2 signaling as a critical driver of TMEM doorway-mediated vascular permeability and metastatic dissemination in breast cancer.

The TGIF1 transcription factor gene is present on chromosome 18, which is subject to whole chromosome copy number reduction in colon cancer. Despite this, TGIF1 expression is significantly higher in cancer than in normal. In mice complete deletion of Tgif1 reduced tumor burden in an Apc mutant model of intestinal cancer. Here we show that reducing TGIF1 expression in a human colon cancer cell line slows proliferation and reduces growth of orthotopic xenografts. To ask if additional genes with copy number loss are more highly expressed in tumors we identified chromosomal regions subject to copy number reductions from ten TCGA cancer datasets. Within these regions a small proportion of genes, generally less than 10%, are expressed at higher levels in the tumor than in corresponding normal samples. Enrichment analysis using a set of 435 genes that have copy number reduction and increased expression identified mitosis as the most enriched gene set and FOXM1 and E2F family transcription factors as potential regulators. For mitotic genes, the average expression increase in tumor compared to normal is independent of copy number. In contrast, while DepMap common essential genes are generally more highly expressed in cancer than normal tissue, the relative increase in expression tracks well with copy number. Similarly, expression differences for gene sets such as S-phase, rRNA processing and DNA repair show increased expression in cancer versus normal, but changes also track with copy number. Thus, genes with increased expression despite copy number reduction may represent the output of key pro-tumorigenic transcriptional programs and could be potential therapeutic targets.

Activating mutations in PI3K are one of the most frequent mutations in breast cancer and are associated with worse patient outcomes in many breast cancer subtypes. Despite intense interest, cancer treatments that target the PI3K pathway have been only modestly effective due to intrinsic and acquired resistance mechanisms which reactivate PI3K signaling. Here, we characterize a feedback mechanism by which PI3K pathway inhibitors increase insulin receptor substrate 2 (IRS2) abundance and demonstrate the role of IRS2 in promoting resistance to these drugs. In PIK3CA mutant breast tumors and cell lines, there is a significant reduction in IRS2 mRNA and protein abundance which is reversed by PI3K pathway inhibition and mediated by the transcription factor FOXO3. PIK3CA mutations do not alter IRS1 expression. IRS2 confers resistance to PI3K pathway inhibition by sustaining PI3K signaling in PIK3CA mutant, but not wild-type breast cancer cells. Increased IRS2 abundance also correlates with PI3K pathway inhibitor resistance across PI3K mutant cancer cell lines from a variety of tissues. The clinical relevance of these findings is highlighted by the frequency of PI3K mutations in cancer and the identification of a new target to address the challenges associated with prior efforts to block the reactivation of PI3K signaling during PI3K inhibition.