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PDF file - 300K, ADAM12 immunolocalization in a subcutanous Lewis Lung tumor. Immunohistochemical staining of ADAM12 in Lewis Lung tumors grown for 15 days in ADAM12+/- mice using A) 2�g/mL affinity purified ADAM12 polyclonal antibody from ProteinTech Group or B) 10 �g/mL normal rabbit IgG fraction (Dako) as primary antibodies. Sections were counterstained with hematoxylin. A and B use identical magnification with scale bar in B = 20�m Detection was performed using the DakoChemMate detection kit (Dako).

PDF file - 213K, A Pan-cytokeratin expression (green) in immortalized PyMT-A12hz and PyMT-A12null tumor cells. Blue represents DAPI staining. Bar = 12 �m. B. Mean (�SD) total tumor burden on FVB/N mice injected subcutaneously in the right flank with PyM-A12hz (n=11) or PyMT-A12null (n=15) tumor cells. *P < 0.05

PDF file - 819K, List of primer sequences for semi-quantitative and quantitative PCR

PDF file - 504K, Response of xenografts of human prostate cancer specimens to IGF-1R inhibition as a function of NRP2 expression

PDF file - 454K, Role of JNK/c-Jun in regulating NRP2 expression in prostate carcinoma cells

PDF file - 1.8MB, Evaluation of insulin signaling in prostate tumors and cell lines as a function of NRP2 expression and role of VEGF in regulation of Bmi-1

We show that the VEGF receptor neuropilin-2 (NRP2) is associated with high-grade, PTEN-null prostate cancer and that its expression in tumor cells is induced by PTEN loss as a consequence of c-Jun activation. VEGF/NRP2 signaling represses insulin-like growth factor-1 receptor (IGF-IR) expression and signaling, and the mechanism involves Bmi-1–mediated transcriptional repression of the IGF-IR. This mechanism has significant functional and therapeutic implications that were evaluated. IGF-IR expression positively correlates with PTEN and inversely correlates with NRP2 in prostate tumors. NRP2 is a robust biomarker for predicting response to IGF-IR therapy because prostate carcinomas that express NRP2 exhibit low levels of IGF-IR. Conversely, targeting NRP2 is only modestly effective because NRP2 inhibition induces compensatory IGF-IR signaling. Inhibition of both NRP2 and IGF-IR, however, completely blocks tumor growth in vivo.Significance: These results identify a causal role for NRP2 and VEGF/NRP2 signaling in the behavior of aggressive prostate cancers by a mechanism that involves regulation of Bmi-1, a transcriptional repressor implicated in the etiology of prostate cancer induced by loss of PTEN function, and the repression of the IGF-IR. The therapeutic implications are significant because combined inhibition of NRP2 and IGF-IR overcomes the resistance induced by targeting each receptor individually. Cancer Discov; 2(10); 906–21. ©2012 AACR.This article is highlighted in the In This Issue feature, p. 857.

PDF file - 2.5MB, Correlation of NRP2 expression in prostate cancer with clinical parameters and expression of VEGFRs

PDF file - 536K, Invasive behavior of cell lines used and assessment of NRP2 expression in prostate glands and cancer

Expression of ADAM12 is low in most normal tissues but is markedly increased in numerous human cancers, including breast carcinomas. We have previously shown that overexpression of ADAM12 accelerates tumor progression in a mouse model of breast cancer (PyMT). In this study, we found that ADAM12 deficiency reduces breast tumor progression in the PyMT model. However, the catalytic activity of ADAM12 seems to be dispensable for its tumor-promoting effect. Interestingly, we show that ADAM12 endogenously expressed in tumor-associated stroma in the PyMT model does not influence tumor progression, but that ADAM12 expression by tumor cells is necessary for tumor progression in these mice. This finding is consistent with our observation that in human breast carcinoma, ADAM12 is almost exclusively located in tumor cells and, only rarely, seen in the tumor-associated stroma. We hypothesized, however, that the tumor-associated stroma may stimulate ADAM12 expression in tumor cells, on the basis of the fact that TGF-β1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-β1 stimulation of ADAM12-negative Lewis lung tumor cells induced ADAM12 synthesis, and growth of these cells in vivo induced more than 200-fold increase in ADAM12 expression. Our observation that ADAM12 expression is significantly higher in the terminal duct lobular units (TDLU) adjacent to human breast carcinoma compared with TDLUs found in normal breast tissue supports our hypothesis that tumor-associated stroma triggers ADAM12 expression. Mol Cancer Res; 9(11); 1449–61. ©2011 AACR.

PDF file - 429K, Whole-mount section used for ADAM12 profiling in TDLUs, ADH, DCIS, and IDC. H&E-stained sections of breast tissue. A, The insert marks a representative example of an area used for the ADAM12 immunostaining presented in Table 1 and Fig. 5. B, TDLUs and ducts co-existing with carcinoma cells. C, An area with ADH. D, An area with DCIS. E, An area with IDC. Scale bar in B = 60 �m, in E = 30 �m. C-E use identical magnification.

PDF file - 484K, Evaluation of the contribution of FOXO1 to NRP2 expression

PDF file - 238K, Stromal ADAM12 does not influence tumor growthA12null tumor cells examined by Western blot. Western blot analysis of ADAM12 expression in PyMT-A12hz, PyMT-A12null, and B16F10 cell lines. G, Mean (�SD) tumor volumes over time in wild-type (n=5), ADAM12+/- (n=11), or ADAM12-/- (n=6) mice on the C57/Bl6 genetic background injected s.c. with B16F10 melanoma cells.

PDF file - 582K, Analysis of NRP2, IGF-1R and p-AKT in consecutive sections of prostate cancer specimens

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As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression.

Supplementary Figure S1 from A Role for ADAM12 in Breast Tumor Progression and Stromal Cell Apoptosis

Cancer stem cells (CSCs) are a crucial therapeutic target because of their role in resistance to chemo- and radiation therapy, metastasis, and tumor recurrence. Differentiation therapy presents a potential strategy for "defanging" CSCs. To date, only a limited number of small-molecule and nanomaterial-based differentiating agents have been identified. We report here the integrated use of nanoparticle engineering and hypothesis-free sensing to identify nanoparticles capable of efficient differentiation of CSCs into non-CSC phenotypes. Using this strategy, we identified a nanoparticle that induces CSC differentiation by increasing intracellular reactive oxygen species levels. Importantly, this unreported phenotype is more susceptible to drug treatment than either CSCs or non-CSCs, demonstrating a potentially powerful strategy for anticancer therapeutics.

Description, A specific cell surface glycan enables the identification of breast cancer stem cells and contributes to their function., We pursued the hypothesis that specific glycans can be used to distinguish breast cancer stem cells (CSCs) and influence their function. Comparison of CSCs and non-CSCs from multiple breast cancer models revealed that CSCs are distinguished by expression of α2,3 sialylated core2 O-linked glycans. We identified a lectin, SLBR-N, which binds to O-linked α2,3 sialic acids, that was able to enrich for CSCs in vitro and in vivo. This O-glycan is expressed on CD44 and promotes its interaction with hyaluronic acid, facilitating CD44 signaling and CSC properties. In contrast, FUT3, which contributes to sialyl Lewis X (sLeX) production, is preferentially expressed in the non-CSC population, and it antagonizes CSC function. Collectively, our data indicate that SLBR-N can be more efficient at enriching for CSCs than CD44 itself because its use avoids the issues of CD44 splicing and glycan status. These data also reveal how differential glycosylation influences CSC fate.

Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6β4 integrin (β4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that β4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgβ4(flox/flox) mice to generate conditional knock-out of β4 in alveolar progenitor cells. These itgβ4(flox/flox) WAP-Cre(+) mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgβ4(flox/flox) WAP-Cre(−) mice, establishing a novel role for the β4 integrin in alveolar progenitor function and alveologenesis.

Understanding how cancer cells resist ferroptosis is a significant problem that impacts ongoing efforts to stimulate ferroptosis as a therapeutic strategy. We reported that prominin2 is induced by ferroptotic stimuli and functions to resist ferroptotic death. Although this finding has significant implications for therapy, specific prominin2 inhibitors are not available. We rationalized that the mechanism by which prominin2 expression is induced by ferroptotic stress could be targeted, expanding the range of options to overcome ferroptosis resistance. Here, we show that that 4‐hydroxynonenal (4HNE), a specific lipid metabolite formed from the products of lipid peroxidation stimulates PROM2 transcription by a mechanism that involves p38 MAP kinase‐mediated activation of HSF1 and HSF1‐dependent transcription of PROM2. HSF1 inhibitors sensitize a wide variety of resistant cancer cells to drugs that induce ferroptosis. Importantly, the combination of a ferroptosis‐inducing drug and an HSF1 inhibitor causes the cytostasis of established tumors in mice, although neither treatment alone is effective. These data reveal a novel approach for the therapeutic induction of ferroptosis in cancer., Stimulating ferroptosis has emerged as a potential therapeutic strategy against cancer. Some tumor cells, however, are resistant to known ferroptosis stimuli. This study identifies mechanisms that contribute to ferroptosis and develops strategies to overcome resistance.

Ferroptosis is an iron dependent form of regulatory cell death caused by the accumulation of lipid peroxides that can be exploited as a therapeutic for carcinomas and possibly other cancers. However, some populations of carcinoma cells resist ferroptosis, especially those with an epithelial phenotype but the mechanisms that promote resistance are poorly understood. We discovered that ferroptosis resistance in triple-negative breast cancer cells (TNBC) with an epithelial phenotype is dependent on expression of the integrin β4 and its ligand laminin 332 (LM332). LM332 and the integrin β4 maintain an epithelial cell state by downregulation of the EMT factor ZEB1. We further demonstrate that repression of ZEB1 by integrin β4 and LM332 promotes YAP/TAZ-mediated transcription of the C2 subunit of LM332, activating a positive feedback loop. Lastly, we observed that disrupting the β4/LM332 signaling axis sensitizes epithelial TNBC cells to ferroptosis by inducing ZEB1 expression. Our findings reveal a novel role for the extracellular matrix in promoting ferroptosis resistance by sustaining an epithelial phenotype and they suggest potential therapeutic strategies for overcoming this resistance. Citation Format: Emmet R. Karner, Hira Lal Goel, Arthur M. Mercurio. Ferroptosis resistance in epithelial populations of triple negative breast cancer is mediated by laminin 332 and integrin beta4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6097.

Introduction: Triple negative breast cancer (TNBC) represents 15% of all breast cancers, and is a notably aggressive subtype that is prone to early recurrence following initial diagnosis with a median survival of only 9 months following recurrence. One of the few treatment options for patients with advanced and metastatic TNBC is platinum-based chemotherapy, however not all tumors respond, and those that do often become refractory during the course of treatment. The cell surface receptor Neuropilin-2 (NRP2), which acts as a co-receptor for vascular endothelial growth factors (VEGFs), has been shown to be both highly expressed and associated with therapeutic resistance in TNBC. NRP2 also serves as a co-receptor for semaphorins, which may play tumor-suppressor functions in breast cancer. We have previously demonstrated that ATYR2810, a highly specific humanized monoclonal antibody which effectively blocks NRP2/VEGF signaling without disrupting NRP2/Semaphorin 3F signaling, down-regulated epithelial-mesenchymal transition (EMT) genes such as ZEB1 and sensitizes TNBC to chemotherapy both in vitro and in vivo. Results: In this study we have further characterized the breast cancer subtypes that are most responsive to ATYR2810 treatment. Using in vitro 3D colony formation assays (CFAs), we have interrogated ~15 breast cancer cell lines covering luminal, HER2+ and various TNBC subtypes, for responsiveness to ATYR2810 treatment in combination with chemotherapy. All responsive cell lines were associated with the more aggressive subtypes of TNBC such as BL2 (basal-like 2), ML (mesenchymal-like) and MSL (mesenchymal-stem like) subtypes. Additionally, we screened five patient derived xenografts (PDXs) and again found that the responsive PDXs were associated with the more aggressive and chemoresistant subtypes of TNBC. To gain insight into the potential mechanism for enhanced chemosensitivity by ATYR2810 treatment, we performed RNAseq on the PDX tumors. Interestingly, the chemokine receptor CXCR4, which is known to promote drug resistance and tumorigenic potential, was found to be significantly downregulated in responder PDX tumors treated with ATYR2810 in combination with cisplatin as compared to those treated with cisplatin alone. Further, gene set enrichment analysis (GSEA) showed that CXCR4 was found in numerous downregulated pathways enriched in the PDX responder tumors treated with ATYR2810/cisplatin, suggesting that CXCR4 may be driving the responder phenotype of the PDXs. Flow cytometry analysis of TNBC cells treated with ATYR2810 monotherapy in vitro resulted in a reduced frequency of cells expressing CXCR4. Furthermore, ATYR2810 monotherapy inhibited spontaneous lung metastasis in experimental models of mesenchymal TNBC. Conclusion: Taken together, our data suggest that ATYR2810 enhances chemosensitivity in highly aggressive TNBC subtypes, and that this response may be mediated through the downregulation of genes known to be associated with aggressive cancer states such as ZEB1 and CXCR4. ATYR2810 may therefore serve as a novel therapeutic agent for the treatment of advanced and metastatic TNBC and potentially other aggressive cancer types. Zhiwen Xu and Alison Barber are co-first authors of this abstract. Citation Format: Zhiwen Xu, Alison Barber, Christoph Burkart, Hira Lal Goel, Justin Rahman, Kristina Hamel, Zachary Fogassy, Lisa Eide, Clara Polizzi, Jasmine Stamps, Sofia Klopp Savino, Luke Burman, Esther Chong, Suzanne Paz, Arthur M. Mercurio, Leslie A. Nangle. ATYR2810, a fully humanized monoclonal antibody targeting the VEGF-NRP2 pathway sensitizes highly aggressive and chemoresistant TNBC subtypes to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB085.

Although the immune checkpoint function of PD-L1 has dominated its study, its cell autonomous functions are now being realized. Here, we report that PD-L1 localizes in retraction fibers and migrasomes at the rear of migrating cells and it facilitates directionally persistent cell migration, a function that underlies tumor invasion. We also demonstrate that PD-L1 interacts with the β4 integrin in retraction fibers and migrasomes and that this integrin contributes to their formation, a process that is dependent on laminin-332. More specifically, our data reveal that PD-L1 senses low membrane tension at the cell rear during migration, forms lipid rafts that contain the β4 integrin and, consequently, localizes this integrin to sites of retraction fiber formation. Depletion of PD-L1, the β4 integrin or chemical disruption of lipid rafts resulted in reduced retraction fiber formation and stalled directional cell migration. Our findings identify a novel cell autonomous function of PD-L1 in promoting the formation of retraction fibers and migrasomes that is critical for regulating directionally persistent cell migration by interacting with the b4 integrin and altering its dynamics.

Although changes in alternative splicing have been observed in cancer, their functional contributions still remain largely unclear. Here we report that splice isoforms of the cancer stem cell (CSC) marker CD44 exhibit strikingly opposite functions in breast cancer. Bioinformatic annotation in patient breast cancer in The Cancer Genome Atlas (TCGA) database reveals that the CD44 standard splice isoform (CD44s) positively associates with the CSC gene signatures, whereas the CD44 variant splice isoforms (CD44v) exhibit an inverse association. We show that CD44s is the predominant isoform expressed in breast CSCs. Elimination of the CD44s isoform impairs CSC traits. Conversely, manipulating the splicing regulator ESRP1 to shift alternative splicing from CD44v to CD44s leads to an induction of CSC properties. We further demonstrate that CD44s activates the PDGFRβ/Stat3 cascade to promote CSC traits. These results reveal CD44 isoform specificity in CSC and non-CSC states and suggest that alternative splicing provides functional gene versatility that is essential for distinct cancer cell states and thus cancer phenotypes.

Ferroptosis is an iron-dependent form of programmed cell death characterized by the accumulation of lipid-targeting reactive oxygen species that kill cells by damaging their plasma membrane. The lipid repair enzyme GSH peroxidase 4 (GPX4) protects against this oxidative damage and enables cells to resist ferroptosis. Recent work has revealed that matrix-detached carcinoma cells can be susceptible to ferroptosis and that they can evade this fate through the signaling properties of the α6β4 integrin, which sustains GPX4 expression. Although these findings on ferroptosis are provocative, they differ from those in previous studies indicating that matrix-detached cells are prone to apoptosis via a process referred to as anoikis. In an effort to reconcile these discrepant findings, here we observed that matrix-detached epithelial and carcinoma cells cluster spontaneously via a mechanism that involves the cell adhesion protein PVRL4 (also known as Nectin-4). We found that this clustering process allows these cells to survive by stimulating a PVRL4/α6β4/Src signaling axis that sustains GPX4 expression and buffers against lipid peroxidation. In the absence of α6β4, PVRL4-mediated clustering induced an increase in lipid peroxidation that was sufficient for triggering ferroptosis. When the clustering was inhibited, single cells did not exhibit a significant increase in lipid peroxidation in the absence of α6β4, and they were more susceptible to apoptosis than to ferroptosis. These results indicate that ferroptosis induction depends on cell clustering in matrix-detached cells that lack α6β4 and imply that the fate of matrix-detached cells can be determined by the state of their cell–cell interactions.

Cancer stem cells (CSCs) contribute to multidrug resistance, tumor recurrence and metastasis, making them prime therapeutic targets. Their ability to differentiate and lose stem cell properties makes them challenging to study. Currently, there is no simple assay that can quickly capture and trace the dynamic phenotypic changes on the CSC surface. Here, we report rapid discrimination of breast CSCs from non-CSCs using a nanoparticle-fluorescent-protein based sensor. This nanosensor was employed to discriminate CSCs from non-CSCs, as well as CSCs that had differentiated in vitro in two breast cancer models. Importantly, the sensor platform could also discriminate CSCs from the bulk population of cells in patient-derived xenografts of human breast cancer. Taken together, the results obtained demonstrate the feasibility of using the nanosensor to phenotype CSCs and monitor their fate. Furthermore, this approach provides a novel area for therapeutic interventions against these challenging targets.

Lipid peroxidation in the plasma membrane can cause ferroptosis, a form of regulated necrosis. Brown et al. show that matrix detachment can induce ferroptosis, and the α6β4 integrin impedes that process by suppressing expression of the proferroptotic enzyme ACSL4., Increases in lipid peroxidation can cause ferroptosis, a form of cell death triggered by inhibition of glutathione peroxidase 4 (GPX4), which catalyzes the reduction of lipid peroxides and is a target of ferroptosis inducers, such as erastin. The α6β4 integrin protects adherent epithelial and carcinoma cells from ferroptosis induced by erastin. In addition, extracellular matrix (ECM) detachment is a physiologic trigger of ferroptosis, which is evaded by α6β4. The mechanism that enables α6β4 to evade ferroptosis involves its ability to protect changes in membrane lipids that are proferroptotic. Specifically, α6β4-mediated activation of Src and STAT3 suppresses expression of ACSL4, an enzyme that enriches membranes with long polyunsaturated fatty acids and is required for ferroptosis. Adherent cells lacking α6β4 require an inducer, such as erastin, to undergo ferroptosis because they sustain GPX4 expression, despite their increase in ACSL4. In contrast, ECM detachment of cells lacking α6β4 is sufficient to trigger ferroptosis because GPX4 is suppressed. This causal link between α6β4 and ferroptosis has implications for cancer biology and therapy.

Barney et al. used biomaterials to uncover a role for fibronectin in preventing breast cancer cell escape from dormancy., Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, nonproliferative state before reactivation and outgrowth. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system with carefully controlled ECM substrates to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle–arrested, and actively proliferating cells. Cell populations capable of entering dormancy formed an organized, fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation, and cancer cell outgrowth after dormancy required MMP-2–mediated fibronectin degradation. We propose this approach as a useful, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, non-proliferative state before reactivation and outgrowth. For a patient, these post-remission tumors are often drug resistant and highly aggressive, resulting in poor prognosis. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created anin vitrocell culture system that combines carefully controlled ECM substrates with nutrient deprivation to observe entranceintoand exitfromdormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle arrested, and actively proliferating cells. Cell populations that endured extended periods of serum-deprivation-induced dormancy formed an organized, fibrillar fibronectin matrix via αvβ3and α5β1integrin adhesion, ROCK-generated tension, and TGFβ2 stimulation. We surmised that the fibronectin matrix was primarily a mediator of cell survival, not proliferation, during the serum-deprivation stress, bacause cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. Given the difficulty of animal models in observing entrance and exit from dormancy in real-time, we propose this approach as a new,in vitromethod to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF–NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF–NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF–NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ–TEAD transcriptional target. We also discovered that VEGF–NRP2–YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF–NRP2 or YAP/TAZ. These findings reveal roles for VEGF–NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF–NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.

The ability to monitor changes in the expression and localization of integrins is essential for understanding their contribution to development, tissue homeostasis and disease. Here, we pioneered the use of Crispr/Cas9 genome editing to tag an allele of the β4 subunit of the α6β4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin β4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin β4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin β4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin β4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin β4 expression in cells proximal to the wound edge was evident, and a population of integrin β4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin β4 dynamics during invasion and metastasis. Moreover, these integrin β4 reporter cells should facilitate studies on the contribution of this integrin to mammary gland biology and cancer. This article has an associated First Person interview with the first author of the paper.

INTRODUCTION: A growing body of evidence suggests that increased expression of the vascular endothelial growth factor (VEGF) co-receptor Neuropilin-2 (NRP2) is associated with aggressive breast cancers and that VEGF/NRP2 signaling contributes to clinical resistance to chemotherapy and tumor recurrence, making NRP2 a promising therapeutic target. A major limitation that has hampered the development of such a therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that specifically block VEGF/NRP2 signaling. aTyr has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of diseases that involve NRP2 signaling. Among them, ATYR2810 has been characterized to bind to the b1 domain of NRP2 that encompasses the VEGF binding sites. It completely blocks the binding of VEGF to NRP2, and VEGF-induced NRP2/VEGFR dimerization. Importantly, ATYR2810 had no effect on Semaphorin 3F (Sema3F) induced NRP2/PlexinA1 dimerization, demonstrating its specificity for blocking the VEGF/NRP2 pathway. We have shown that ATYR2810, but not a Sema3F-blocking mAb has tumor-inhibitory effects on triple negative breast cancer (TNBC) cell lines or patient-derived organoids. RESULTS: We further evaluated the efficacy of ATYR2810 in combination with standard-of-care therapeutics including Cisplatin and Bevacizumab (anti-VEGF-A blocking antibody). In in vitro 3D methylcellulose colony formation assays, ATYR2810 sensitized TNBC cells to Cisplatin or Bevacizumab and considerably reduced colony formation in combination with these treatments. In an in vivo TNBC xenograft cancer model (MDA-MB-231), ATYR2810 augmented the anti-tumor effects of Cisplatin or Bevacizumab. To explore the underlying molecular mechanism, we performed gene expression profiling with samples treated by ATYR2810 alone and the combo therapy with Cisplatin or Bevacizumab. A number of gene markers of cancer stem cells (CSC) and/or epithelial-mesenchymal transition (EMT) were found to be down-regulated by ATYR2810 treatment in TNBC patient-derived organoids, including a key EMT transcription factor ZEB1. We also confirmed the reduction of ZEB1 protein expression by ATYR2810 treatment in TNBC cells. CONCLUSIONS: These results demonstrate the efficacy of ATYR2810 in combination with standard-of-care therapeutics in in vitro and in vivo TNBC models, and suggest its activity is mediated through inhibiting both EMT and cellular dedifferentiation that renders tumors more sensitive to the treatment regimes. The targeting of VEGF/NRP2 signaling by ATYR2810 may provide a new therapeutic option, and lead to the identification of new treatment biomarkers, which could offer improved efficacy and reduced toxicity in aggressive breast cancers. Citation Format: Zhiwen Xu, Christoph Burkart, Hira L. Goel, Justin Rahman, Clara Polizzi, Matt Seikkula, Luke Burman, Arthur M. Mercurio, Leslie A. Nangle. A domain-specific antibody to NRP2 down-regulated epithelial-mesenchymal transition genes and enhanced efficacy of standard-of-care therapeutics for aggressive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB095.

Lung cancer remains one of the leading cancer types and is the foremost cause of cancer-related deaths, yet treatment options for advanced lung cancer remain limited. Novel therapies targeting factors critical to the progression of lung cancer are essential. Neuropilin-2 (NRP2) acts as a co-receptor for VEGF and has been correlated with aggressive cancer growth and metastasis. The expression of NRP2 has been shown to be upregulated in several cancers, including non-small cell lung cancer (NSCLC), and the expression of NRP2 in NSCLC is associated with increased invasive tumor growth in vitro as well as a significant decrease in patient survival. As shown previously, aTyr has developed ATYR2810, a domain-specific anti-human monoclonal antibody to NRP2 which blocks its binding to VEGF. Importantly, ATYR2810 blocks VEGF binding in a highly specific manner and does not affect the binding of Semaphorin-3F or subsequent Semaphorin-3F-induced dimerization of NRP2 and PlexinA1. In this study, we have used ATYR2810 to examine the effects of blocking VEGF-mediated NRP2 signaling both as monotherapy and in combination therapy. Using in vitro 3D colony formation assays, we found that ATYR2810 used as monotherapy led to a significant reduction in colony formation in the A549 NSCLC cell line. The effects of ATYR2810 were also examined in NSCLC xenografts, where the use of ATYR2810 as a single agent was found to result in the inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ATYR2810 as monotherapy was comparable to that of cisplatin monotherapy, suggesting that ATYR2810 may be a potent inhibitor of tumor growth in the A549 xenograft model. The effects of ATYR2810 combination therapy in NSCLC xenografts was also examined, and we found that the use of ATYR2810 in combination with either 5-FU or cisplatin enhanced tumor growth inhibition as compared to the use of either chemotherapeutic agent alone. Taken together, these results suggest that NRP2 may be an important target in aggressive NSCLC and that the use of our novel ATYR2810 antibody to block VEGF-mediated NRP2 signaling may serve as a valuable therapeutic agent both as monotherapy as well as in combination therapy. Citation Format: Alison G. Barber, Zhiwen Xu, Justin Rahman, Hira Lal Goel, Arthur M. Mercurio, Christoph Burkart, Leslie A. Nangle. The Neuropilin-2 targeting antibody ATYR2810 inhibits non-small cell lung cancer tumor growth in monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB234.

SummaryAutocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTENpc−/− transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity of prostate tumors to bevacizumab. These data reveal that prostate tumors harbor cells with stem cell properties that are resistant to inhibitors of VEGF/VEGFR signaling. Combining the use of available VEGF/VEGFR-targeted therapies with P-Rex1 or Rac1 inhibition should improve the efficacy of these therapies significantly.

Introduction: There is a strong body of evidence indicating that the expression of Neuropilin-2 (NRP2) is enriched in breast cancer stem cells (CSCs) and that NRP2 signaling is critical for breast CSC function and resistance development. For this reason, the rationale for targeting NRP2 as a therapeutic strategy is compelling and timely. A major limitation that has hampered the development of this therapy, however, has been the lack of availability of high-quality anti-human NRP2 monoclonal antibodies (mAbs) that block NRP2 signalling. aTyr Pharma has generated a panel of high-quality, anti-human NRP2 mAbs that have the potential to be developed for the clinical management of breast cancer. A significant advance made by aTyr is that through specific domain reactivity, they have demonstrated differential effects on ligand blocking, receptor homo and heterodimerization and functional activity. Importantly a subset of such antibodies show differential activity in the mammosphere assay of triple negative breast cancer. Results: Flow cytometry was used to assess the specificity of the in-house anti-NRP2 mAbs to NRP2 using A549 wild type versus NRP2 knockout clonal cells. The in-house anti-NRP2 mAbs bound to A549 wild-type cells while showing little or no binding to the NRP2 knockout clonal cells, and exhibited significantly superior specificity, and sensitivity compared to existing commercially available antibodies. Displacement studies demonstrated that the tested anti-NRP2 mAbs showed different capabilities in blocking of VEGF-C or SEMA-3F binding to Expi293-hNRP2 cells, and were categorized as blockers (>90% inhibition), partial blockers (30-90% inhibition), or non-blockers (no obvious inhibition). To further extend the assessment of the biological activity of the anti-NRP2 antibodies, their activity was assessed in a receptor dimerization assay. Vectors encoding a split luciferase pBiT1.1 and pBiT2.1, and a cell permeable substrate, were obtained from Promega corporation. The complete extracellular domain and transmembrane helices of NRP2, FLT4 (VEGFR3), KDR (VEGFR2) and plexin A1 (PLXNA1) were cloned into the vectors and screened for optimal orientation, following established methods. Tested antibodies were able to impair respective VEGF and SEMA3 induced dimerization of receptor pairs NRP2/FLT4, NRP2/KDR and NRP2/PLXNA1. Surprisingly select antibodies show extremely specific and non-obvious functional differentiation. Direct functional assessment of a subset of these antibodies on breast CSCs revealed that the VEGF blocking, but not the SEMA3 blocking anti-NRP2 amAbs had the ability to inhibit serial passage mammosphere formation, an indicator of self-renewal potential. Conclusions: A series of domain specific antibodies to NRP2 have been developed and characterized. These antibodies show differential binding to specific domains of NRP2, can inhibit either VEGF-C or Sema3F binding to NRP2, and differentially effect receptor dimerization. The use of these antibodies enabled us to implicate VEGF-C/NRP2 signaling but not SEMA-3F/NRP2 signaling in the function of breast CSCs. Citation Format: Leslie A. Nangle, Luke Burman, Hira L. Goel, Zhiwen Xu, Kristina Hamel, Nathaniel Bloom, Arthur M. Mercurio. Domain-specific antibodies to Neuropilin 2 implicate VEGF-C and not Semaphorin 3F in breast cancer stem cell function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1785.

Ferroptosis, regulated cell death characterized by the iron-dependent accumulation of lethal lipid reactive oxygen species, contributes to tissue homeostasis and numerous pathologies, and it may be exploited for therapy. Cells differ in their sensitivity to ferroptosis, however, and a key challenge is to understand mechanisms that contribute to resistance. Using RNA-Seq to identify genes that contribute to ferroptosis resistance, we discovered that pro-ferroptotic stimuli including inhibition of the lipid hydroperoxidase GPX4 and detachment from the extracellular matrix induce expression of prominin2, a pentaspanin protein implicated in regulation of lipid dynamics. Prominin2 facilitates ferroptosis resistance in mammary epithelial and breast carcinoma cells. Mechanistically, prominin2 promotes the formation of ferritin-containing multi-vesicular bodies (MVBs) and exosomes that transport iron out of the cell, inhibiting ferroptosis. These findings reveal that ferroptosis resistance can be driven by a prominin2-MVB/exosome-ferritin pathway and they have broad implications for iron homeostasis, intracellular trafficking, and cancer.

The function of vascular endothelial growth factor (VEGF) in cancer extends beyond angiogenesis and vascular permeability. Specifically, VEGF-mediated signaling occurs in tumor cells and this signaling contributes to key aspects of tumorigenesis including the self-renewal and survival of cancer stem cells (CSCs). In addition to VEGF receptor tyrosine kinases, the neuropilins (NRPs) are critical for mediating the effects of VEGF on CSCs, primarily because of their ability to impact the function of growth factor receptors and integrins. VEGF/NRP signaling can regulate the expression and function of key molecules that have been implicated in CSC function including Rho family guanosine triphosphatases (GTPases) and transcription factors. The VEGF/NRP signaling axis is a prime target for therapy because it can confer resistance to standard chemotherapy, which is ineffective against most CSCs. Indeed, several studies have shown that targeting either NRP1 or NRP2 can inhibit tumor initiation and decrease resistance to other therapies.

Understanding how cells resist ferroptosis is necessary for exploiting this iron-dependent mode of cell death for the treatment of cancer and other diseases. We discovered that cells resist ferroptosis by enabling a PROMININ2-dependent iron export pathway involving multivesicular body/exosome trafficking of iron out of the cell, diminishing the intracellular iron needed for ferroptosis.