YAP/TAZ-mediated regulation of laminin 332 is enabled by ß4 integrin repression of ZEB1 to promote ferroptosis resistance.

We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin ß4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the ß4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the ß4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of ß4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of ß4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the ß4 integrin.

The calcium channel TRPC6 promotes chemotherapy-induced persistence by regulating integrin α6 mRNA splicing.

Understanding the cell biological mechanisms that enable tumor cells to persist after therapy is necessary to improve the treatment of recurrent disease. Here, we demonstrate that transient receptor potential channel 6 (TRPC6), a channel that mediates calcium entry, contributes to the properties of breast cancer stem cells, including resistance to chemotherapy, and that tumor cells that persist after therapy are dependent on TRPC6. The mechanism involves the ability of TRPC6 to regulate integrin α6 mRNA splicing. Specifically, TRPC6-mediated calcium entry represses the epithelial splicing factor ESRP1 (epithelial splicing regulatory protein 1), which enables expression of the integrin α6B splice variant. TRPC6 and α6B function in tandem to facilitate stemness and persistence by activating TAZ and, consequently, repressing Myc. Therapeutic inhibition of TRPC6 sensitizes triple-negative breast cancer (TNBC) cells and tumors to chemotherapy by targeting the splicing of α6 integrin mRNA and inducing Myc. These data reveal a Ca2+-dependent mechanism of chemotherapy-induced persistence, which is amenable to therapy, that involves integrin mRNA splicing.

Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells.

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.

Therapeutic blocking of VEGF binding to neuropilin-2 diminishes PD-L1 expression to activate antitumor immunity in prostate cancer.

Prostate cancers are largely unresponsive to immune checkpoint inhibitors (ICIs), and there is strong evidence that programmed death-ligand 1 (PD-L1) expression itself must be inhibited to activate antitumor immunity. Here, we report that neuropilin-2 (NRP2), which functions as a vascular endothelial growth factor (VEGF) receptor on tumor cells, is an attractive target to activate antitumor immunity in prostate cancer because VEGF-NRP2 signaling sustains PD-L1 expression. NRP2 depletion increased T cell activation in vitro. In a syngeneic model of prostate cancer that is resistant to ICI, inhibition of the binding of VEGF to NRP2 using a mouse-specific anti-NRP2 monoclonal antibody (mAb) resulted in necrosis and tumor regression compared with both an anti-PD-L1 mAb and control immunoglobulin G. This therapy also decreased tumor PD-L1 expression and increased immune cell infiltration. We observed that the NRP2, VEGFA, and VEGFC genes are amplified in metastatic castration-resistant and neuroendocrine prostate cancer. We also found that individuals with NRP2High PD-L1High metastatic tumors had lower androgen receptor expression and higher neuroendocrine prostate cancer scores than other individuals with prostate cancer. In organoids derived from patients with neuroendocrine prostate cancer, therapeutic inhibition of VEGF binding to NRP2 using a high-affinity humanized mAb suitable for clinical use also diminished PD-L1 expression and caused a substantial increase in immune-mediated tumor cell killing, consistent with the animal studies. These findings provide justification for the initiation of clinical trials using this function-blocking NRP2 mAb in prostate cancer, especially for patients with aggressive disease.

Inhibition of VEGF binding to neuropilin-2 enhances chemosensitivity and inhibits metastasis in triple-negative breast cancer.

Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.

O-linked α2,3 sialylation defines stem cell populations in breast cancer.

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.

Alveolar progenitor cells in the mammary gland are dependent on the ß4 integrin.

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 α6ß4 integrin (ß4) 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 ß4 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 itgß4flox/flox mice to generate conditional knock-out of ß4 in alveolar progenitor cells. These itgß4flox/floxWAP-Cre+ mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgß4flox/floxWAP-Cre- mice, establishing a novel role for the ß4 integrin in alveolar progenitor function and alveologenesis.

Real-time imaging of integrin ß4 dynamics using a reporter cell line generated by Crispr/Cas9 genome editing.

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 ß4 subunit of the α6ß4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin ß4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin ß4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin ß4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin ß4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin ß4 expression in cells proximal to the wound edge was evident, and a population of integrin ß4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin ß4 dynamics during invasion and metastasis. Moreover, these integrin ß4 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.

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression.

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.

CD44 splice isoform switching determines breast cancer stem cell state.

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 PDGFRß/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.

αvß3 Integrin Mediates Radioresistance of Prostate Cancer Cells through Regulation of Survivin.

The αvß3 integrin is involved in various physiologic and pathologic processes such as wound healing, angiogenesis, tumor growth, and metastasis. The impact of αvß3 integrin on the radiosensitivity of prostate cancer cells and the molecular mechanism controlling cell survival in response to ionizing radiation (IR) was investigated. Both LNCaP cells stably transfected with αvß3 integrin and PC-3 cells that contain endogenous ß3 integrin were used. This study demonstrated that αvß3 integrin increases survival of αvß3-LNCaP cells upon IR while small hairpin RNA (shRNA)-mediated knockdown of αvß3 integrin in PC-3 cells sensitizes to radiation. Expression of αvß3 integrin in LNCaP cells also enhances anchorage-independent cell growth while knockdown of αvß3 integrin in PC-3 cells inhibits anchorage-independent cell growth. The αvß3 antagonist, cRGD, significantly increases radiosensitivity in both αvß3-LNCaP and PC-3 cells. Moreover, αvß3 integrin prevents radiation-induced downregulation of survivin. Inhibition of survivin expression by siRNA or shRNA enhances IR-induced inhibition of anchorage-independent cell growth. Overexpression of wild-type survivin in PC-3 cells treated with αvß3 integrin shRNA increases survival of cells upon IR. These findings reveal that αvß3 integrin promotes radioresistance and regulates survivin levels in response to IR. IMPLICATIONS: Future translational research on targeting αvß3 integrin and survivin may reveal novel approaches as an adjunct to radiotherapy for patients with prostate cancer.

VEGF-neuropilin-2 signaling promotes stem-like traits in breast cancer cells by TAZ-mediated repression of the Rac GAP ß2-chimaerin.

The role of vascular endothelial growth factor (VEGF) signaling in cancer is not only well known in the context of angiogenesis but also important in the functional regulation of tumor cells. Autocrine VEGF signaling mediated by its co-receptors called neuropilins (NRPs) appears to be essential for sustaining the proliferation and survival of cancer stem cells (CSCs), which are implicated in mediating tumor growth, progression, and drug resistance. Therefore, understanding the mechanisms involved in VEGF-mediated support of CSCs is critical to successfully treating cancer patients. The expression of the Hippo effector TAZ is associated with breast CSCs and confers stem cell-like properties. We found that VEGF-NRP2 signaling contributed to the activation of TAZ in various breast cancer cells, which mediated a positive feedback loop that promoted mammosphere formation. VEGF-NRP2 signaling activated the GTPase Rac1, which inhibited the Hippo kinase LATS, thus leading to TAZ activity. In a complex with the transcription factor TEAD, TAZ then bound and repressed the promoter of the gene encoding the Rac GTPase-activating protein (Rac GAP) ß2-chimaerin. By activating GTP hydrolysis, Rac GAPs effectively turn off Rac signaling; hence, the TAZ-mediated repression of ß2-chimaerin resulted in sustained Rac1 activity in CSCs. Depletion of ß2-chimaerin in non-CSCs increased Rac1 activity, TAZ abundance, and mammosphere formation. Analysis of a breast cancer patient database revealed an inverse correlation between ß2-chimaerin and TAZ expression in tumors. Our findings highlight an unexpected role for ß2-chimaerin in a feed-forward loop of TAZ activation and the acquisition of CSC properties.

The α6ß4 integrin promotes resistance to ferroptosis.

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 α6ß4 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 α6ß4. The mechanism that enables α6ß4 to evade ferroptosis involves its ability to protect changes in membrane lipids that are proferroptotic. Specifically, α6ß4-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 α6ß4 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 α6ß4 is sufficient to trigger ferroptosis because GPX4 is suppressed. This causal link between α6ß4 and ferroptosis has implications for cancer biology and therapy.

P-Rex1 Promotes Resistance to VEGF/VEGFR-Targeted Therapy in Prostate Cancer.

Autocrine 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 PTEN(pc-/-) 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.

Structural basis for VEGF-C binding to neuropilin-2 and sequestration by a soluble splice form.

Vascular endothelial growth factor C (VEGF-C) is a potent lymphangiogenic cytokine that signals via the coordinated action of two cell surface receptors, Neuropilin-2 (Nrp2) and VEGFR-3. Diseases associated with both loss and gain of VEGF-C function, lymphedema and cancer, respectively, motivate studies of VEGF-C/Nrp2 binding and inhibition. Here, we demonstrate that VEGF-C binding to Nrp2 is regulated by C-terminal proteolytic maturation. The structure of the VEGF-C C terminus in complex with the ligand binding domains of Nrp2 demonstrates that a cryptic Nrp2 binding motif is released upon proteolysis, allowing specific engagement with the b1 domain of Nrp2. Based on the identified structural requirements for Nrp2 binding to VEGF-C, we hypothesized that the endogenous secreted splice form of Nrp2, s9Nrp2, may function as a selective inhibitor of VEGF-C. We find that s9Nrp2 forms a stable dimer that potently inhibits VEGF-C/Nrp2 binding and cellular signaling. These data provide critical insight into VEGF-C/Nrp2 binding and inhibition.

A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bß1 integrin to sustain breast cancer stem cells.

Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bß1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

Regulated splicing of the α6 integrin cytoplasmic domain determines the fate of breast cancer stem cells.

Although the α6ß1 integrin has been implicated in the function of breast and other cancer stem cells (CSCs), little is known about its regulation and relationship to mechanisms involved in the genesis of CSCs. We report that a CD44(high)/CD24(low) population, enriched for CSCs, is comprised of distinct epithelial and mesenchymal populations that differ in expression of the two α6 cytoplasmic domain splice variants: α6A and α6B. α6Bß1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by α6A integrins. The generation of α6Bß1 is tightly controlled and occurs as a consequence of an autocrine vascular endothelial growth factor (VEGF) signaling that culminates in the transcriptional repression of a key RNA-splicing factor. These data alter our understanding of how α6ß1 contributes to breast cancer, and they resolve ambiguities regarding the use of total α6 (CD49f) expression as a biomarker for CSCs.

VEGF targets the tumour cell.

The function of vascular endothelial growth factor (VEGF) in cancer is not limited to angiogenesis and vascular permeability. VEGF-mediated signalling occurs in tumour cells, and this signalling contributes to key aspects of tumorigenesis, including the function of cancer stem cells and tumour initiation. In addition to VEGF receptor tyrosine kinases, the neuropilins are crucial for mediating the effects of VEGF on tumour cells, primarily because of their ability to regulate the function and the trafficking of growth factor receptors and integrins. This has important implications for our understanding of tumour biology and for the development of more effective therapeutic approaches.

GLI1 regulates a novel neuropilin-2/α6ß1 integrin based autocrine pathway that contributes to breast cancer initiation.

The characterization of cells with tumour initiating potential is significant for advancing our understanding of cancer and improving therapy. Aggressive, triple-negative breast cancers (TNBCs) are enriched for tumour-initiating cells (TICs). We investigated that hypothesis that VEGF receptors expressed on TNBC cells mediate autocrine signalling that contributes to tumour initiation. We discovered the VEGF receptor neuropilin-2 (NRP2) is expressed preferentially on TICs, involved in the genesis of TNBCs and necessary for tumour initiation. The mechanism by which NRP2 signalling promotes tumour initiation involves stimulation of the α6ß1 integrin, focal adhesion kinase-mediated activation of Ras/MEK signalling and consequent expression of the Hedgehog effector GLI1. GLI1 also induces BMI-1, a key stem cell factor, and it enhances NRP2 expression and the function of α6ß1, establishing an autocrine loop. NRP2 can be targeted in vivo to retard tumour initiation. These findings reveal a novel autocrine pathway involving VEGF/NRP2, α6ß1 and GLI1 that contributes to the initiation of TNBC. They also support the feasibility of NRP2-based therapy for the treatment of TNBC that targets and impedes the function of TICs.