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.

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.

Id2 complexes with the SNAG domain of Snai1 inhibiting Snai1-mediated repression of integrin ß4.

The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as α6ß4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits ß4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress ß4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the ß4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated ß4 repression with a concomitant increase in H3K27Me3 modification on the ß4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression.

Estrogen receptor ß sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription.

Estrogen receptor ß (ERß) promotes the degradation of hypoxia inducible factor 1α (HIF-1α), which contributes to the ability of this hormone receptor to sustain the differentiation of epithelial and carcinoma cells. Although the loss of ERß and consequent HIF-1 activation occur in prostate cancer with profound consequences, the mechanism by which ERß promotes the degradation of HIF-1α is unknown. We report that ERß regulates the ligand (3ß-adiol)-dependent transcription of prolyl hydroxylase 2 (PHD2) also known as Egl nine homolog 1 (EGLN1), a 2-oxoglutarate-dependent dioxygenase that hydroxylates HIF-1α and targets it for recognition by the von Hippel-Lindau tumor suppressor and consequent degradation. ERß promotes PHD2 transcription by interacting with a unique estrogen response element in the 5' UTR of the PHD2 gene that functions as an enhancer. PHD2 itself is critical for maintaining epithelial differentiation. Loss of PHD2 expression or inhibition of its function results in dedifferentiation with characteristics of an epithelial-mesenchymal transition, and exogenous PHD2 expression in dedifferentiated cells can restore an epithelial phenotype. Moreover, expression of HIF-1α in cells that express PHD2 does not induce dedifferentiation but expression of HIF-1α containing mutations in the proline residues that are hydroxylated by PHD2 induces dedifferentiation. These data describe a unique mechanism for the regulation of HIF-1α stability that involves ERß-mediated transcriptional regulation of PHD2 and they highlight an unexpected role for PHD2 in maintaining epithelial differentiation.

IMP3 protein promotes chemoresistance in breast cancer cells by regulating breast cancer resistance protein (ABCG2) expression.

IMP3, a member of a family of insulin-like growth factor II (IGF-II) mRNA-binding proteins (IMPs), is expressed preferentially in triple-negative breast cancers, which are resistant to many chemotherapeutics. However, the mechanisms by which it impacts breast cancer have not been elucidated. We hypothesized a role for IMP3 in chemoresistance based on these observations. Depletion of IMP3 expression in triple-negative breast cancer cells increased their sensitivity to doxorubicin and mitoxantrone significantly but not to taxol. Given that doxorubicin and mitoxantrone are effluxed by breast cancer resistance protein (BCRP), we assessed whether IMP3 regulates BCRP. The data obtained demonstrate that IMP3 binds to BCRP mRNA and regulates BCRP expression. These findings are significant because they provide insight into the mechanism by which IMP3 contributes to aggressive cancers, and they highlight the potential for targeting this mRNA-binding protein for the clinical management of cancer.

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.

VEGF/neuropilin-2 regulation of Bmi-1 and consequent repression of IGF-IR define a novel mechanism of aggressive prostate cancer.

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.

Neuropilin-2 regulates α6ß1 integrin in the formation of focal adhesions and signaling.

The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the α6ß1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6ß1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6ß1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6ß1 integrin with the cytoskeleton. NRP2 also facilitates α6ß1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6ß1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the α6ß1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.

Neuropilin-2 promotes branching morphogenesis in the mouse mammary gland.

Although the neuropilins were characterized as semaphorin receptors that regulate axon guidance, they also function as vascular endothelial growth factor (VEGF) receptors and contribute to the development of other tissues. Here, we assessed the role of NRP2 in mouse mammary gland development based on our observation that NRP2 is expressed preferentially in the terminal end buds of developing glands. A floxed NRP2 mouse was bred with an MMTV-Cre strain to generate a mammary gland-specific knockout of NRP2. MMTV-Cre;NRP2(loxP/loxP) mice exhibited significant defects in branching morphogenesis and ductal outgrowth compared with either littermate MMTV-Cre;NRP2(+/loxP) or MMTV-Cre mice. Mechanistic insight into this morphological defect was obtained from a mouse mammary cell line in which we observed that VEGF(165), an NRP2 ligand, induces branching morphogenesis in 3D cultures and that branching is dependent upon NRP2 as shown using shRNAs and a function-blocking antibody. Epithelial cells in the mouse mammary gland express VEGF, supporting the hypothesis that this NRP2 ligand contributes to mammary gland morphogenesis. Importantly, we demonstrate that VEGF and NRP2 activate focal adhesion kinase (FAK) and promote FAK-dependent branching morphogenesis in vitro. The significance of this mechanism is substantiated by our finding that FAK activation is diminished significantly in developing MMTV-Cre;NRP2(loxP/loxP) mammary glands compared with control glands. Together, our data reveal a VEGF/NRP2/FAK signaling axis that is important for branching morphogenesis and mammary gland development. In a broader context, our data support an emerging hypothesis that directional outgrowth and branching morphogenesis in a variety of tissues are influenced by signals that were identified initially for their role in axon guidance.

miR-10b targets Tiam1: implications for Rac activation and carcinoma migration.

Understanding the mechanisms by which specific microRNAs regulate cell migration and invasion is a timely and significant problem in cancer cell biology. miR-10b is of interest in this regard because its expression is altered in breast and other cancers. Our analysis of potential miR-10b targets identified Tiam1 (T lymphoma invasion and metastasis 1), a guanidine exchange factor for Rac. We demonstrate, using an miR-10b synthetic precursor, expression vector, and antisense oligonucleotide, that miR-10b represses Tiam1 expression in breast carcinoma cells and that it interacts with the 3'-UTR of Tiam1. Consistent with the involvement of Tiam1 in cell motility, we observed that miR-10b suppresses the ability of breast carcinoma cells to migrate and invade. Importantly, we demonstrate that miR-10b also inhibits Tiam1-mediated Rac activation. These data provide a mechanism for the regulation of Tiam1-mediated Rac activation in breast cancer cells and need to be considered in the context of other reported functions for miR-10b.

ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading.

High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor beta (ERbeta) expression. We report that a key function of ERbeta and its specific ligand 5alpha-androstane-3beta,17beta-diol (3beta-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-beta and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERbeta expression, and loss of ERbeta is sufficient to promote an EMT. The mechanism involves ERbeta-mediated destabilization of HIF-1alpha and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1alpha and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.

Regulation of beta 4-integrin expression by epigenetic modifications in the mammary gland and during the epithelial-to-mesenchymal transition.

The beta 4 integrin is expressed in epithelial cells, a few other cell types and in some carcinomas. Despite this restricted expression pattern and the functional importance of beta 4 integrin in epithelial and carcinoma biology, little is known about how its expression is regulated. Here, we assessed the epigenetic regulation of beta 4 integrin based on the presence of a large CpG island in the beta 4-integrin gene promoter. We separated basal (beta 4+) and luminal (beta 4-) epithelial cells from the mammary glands of K14-eGFP mice and demonstrated that the beta 4-integrin promoter is unmethylated in basal cells and methylated in luminal cells. We also observed that expression of beta 4 integrin and E-cadherin is lost during the epithelial-to-mesenchymal transition (EMT) of mammary gland cells induced by transforming growth factor beta (TGFbeta), which is coincident with de novo DNA methylation, a decrease in active histone modifications (H3K9Ac and H3K4me3) and an increase in the repressive histone modification H3K27me3. Furthermore, TGFbeta withdrawal promotes a mesenchymal-to-epithelial transition (MET) and triggers the re-expression of beta 4 integrin and E-cadherin. Intriguingly, demethylation at either promoter is not obligatory for transcriptional reactivation after TGFbeta withdrawal. However, both H3K9Ac and H3K4me3 modifications are restored during the MET, and H3K27me3 is reduced, strongly suggesting that reversible histone modifications rather than DNA demethylation are the predominant factors in reactivating expression of these genes. Our data indicate that complex epigenetic modifications contribute to the regulation of the beta 4 integrin and E-cadherin.

Epithelial-mesenchymal transition and colorectal cancer: gaining insights into tumor progression using LIM 1863 cells.

In addition to allowing epithelial cells to escape the structural constraints imposed by tissue architecture and adopt a phenotype more amenable to cell movement, it is now recognized that the epithelial-mesenchymal transition (EMT) may also represent a critical component permitting the progression of carcinomas towards invasive and metastatic disease. However, data supporting the actual occurrence of EMT in specific solid tumors and its relevance to the process of progression of these cancers has been scant. Despite an extensive knowledge of the genetic basis for colorectal cancer, the translation of this information into effective treatments has been limited. Clearly, there is a desperate need for new and improved therapies and since the switch to a metastatic phenotype is critical for outcome, it is of paramount importance to elucidate the biology that underlies the progression of this disease. Thus, the unique LIM 1863 model for studying the EMT of colorectal carcinoma has been used to both substantiate the importance of the transition for this cancer type and to identify molecular events that contribute to disease progression. Importantly, it has emerged that not only does EMT enhance migratory capacity, but also elicits additional selective advantages to colonic tumor cells. Specifically, the acquisition of autocrine growth factor signaling loops, mechanisms to evade apoptosis, and expression of specific integrins allowing invasive cells to interact with interstitial matrices and sustain activation of TGF-beta combine to provide a compelling new biochemical framework for understanding how EMT contributes to tumor evolution.