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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.

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.

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.

Summary: Extracellular vesicles (EVs) play key roles in transporting key molecular constituents as cargo for extracellular trafficking. While several approaches have been developed to extract EVs from mammalian cells, the specific method of EV isolation can have a profound effect on membrane integrity and yield. Here, we describe a step-by-step procedure to separate EVs from adherent epithelial cells using differential ultracentrifugation. Separated EVs can be further analyzed by immunoblotting, mass spectrometry, and transmission electron microscopy to derive EV yield and morphology.For complete details on the use and execution of this protocol, please refer to Brown et al. (2019).

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.

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.

SummaryIMP3 is a fetal protein not expressed in normal adult tissues. IMP3 is an oncoprotein and a useful biomarker for a variety of malignancies and is associated with reduced overall survival of a number of them. IMP3 expression and its prognostic value for patients with intrahepatic cholangiocarcinoma (ICC) have not been well investigated. The molecular mechanism underlying IMP3 expression in human cancer cells remains to be elucidated. Here we investigated IMP3 expression in ICC and adjacent nonneoplastic liver in 72 unifocal primary ICCs from a single institute by immunohistochemistry, immunoblotting, and real-time polymerase chain reaction. IMP3 was specifically expressed in cancer cells but not in the surrounding normal tissue, and 59 (82%) of 72 ICCs were IMP3 positive by immunohistochemistry. Among 35 cases with lymphovascular invasion, 26 (74%) showed IMP3 positivity in lymph node metastases. IMP3 expression was significantly correlated with tumor size, pathological grade, metastasis, and clinical stage. Kaplan-Meier analysis demonstrated an inverse correlation between IMP3 expression and overall survival rate. Multivariate analysis revealed that IMP3 was the only risk factor associated with survival. To further explore the mechanism of IMP3 expression in cancers, we identified 2 CpG islands at IMP3 proximal promoter. Interestingly, the IMP3 promoter was almost completely demethylated in ICCs in contrast to densely methylated promoter in normal liver tissues. IMP3 expression is a useful biomarker for ICCs and can provide an independent prognostic value for patients with ICC. To our knoweldge, this is the first direct evidence of epigenetic deregulation of IMP3 in human cancer.

Summary Although the α6β1 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. α6Bβ1 expression defines the mesenchymal population and is necessary for CSC function, a function that cannot be executed by α6A integrins. The generation of α6Bβ1 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 α6β1 contributes to breast cancer, and they resolve ambiguities regarding the use of total α6 (CD49f) expression as a biomarker for CSCs.

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 multivesicular 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 have broad implications for iron homeostasis, intracellular trafficking, and cancer.

SummaryThe role of ERβ in prostate cancer is unclear, although loss of ERβ is associated with aggressive disease. Given that mice deficient in ERβ do not develop prostate cancer, we hypothesized that ERβ loss occurs as a consequence of tumorigenesis caused by other oncogenic mechanisms and that its loss is necessary for tumorigenesis. In support of this hypothesis, we found that ERβ is targeted for repression in prostate cancer caused by PTEN deletion and that loss of ERβ is important for tumor formation. ERβ transcription is repressed by BMI-1, which is induced by PTEN deletion and important for prostate tumorigenesis. This finding provides a mechanism for how ERβ expression is regulated in prostate cancer. Repression of ERβ contributes to tumorigenesis because it enables HIF-1/VEGF signaling that sustains BMI-1 expression. These data reveal a positive feedback loop that is activated in response to PTEN loss and sustains BMI-1.

In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), while extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12 which encodes the ubiquitin ligase Rnf12/RLIM. Here we find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy. Alveolar cells lacking Rnf12/RLIM undergo apoptosis as soon as they differentiate from mammary epithelia. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to non-random XCI in mammary epithelial cells which primarily silence their maternal X chromosomes. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and provide strong evidence for an imprinted XCI pattern in mammary epithelial cells opposite to that found in extraembryonic trophoblast cells.

The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.

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.

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.

Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-kappaB, which in turn leads to increased fibronectin gene expression, signaling by beta1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer.

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptor molecules that function as signaling intermediates downstream of activated cell surface receptors. Based on data implicating IRS-2 but not IRS-1 in breast cancer invasion, survival, and metastasis, we assessed the contribution of IRS-1 and IRS-2 to aerobic glycolysis, which is known to impact tumor growth and progression. For this purpose, we used tumor cell lines derived from transgenic mice that express the polyoma virus middle T antigen (PyV-MT) in the mammary gland and that are wild-type (WT) or null for either Irs-1 (Irs-1–/–) or Irs-2 (Irs-2–/–). Aerobic glycolysis, as assessed by the rate of lactic acid production and glucose consumption, was diminished significantly in Irs-2–/– cells when compared with WT and Irs-1–/– cells. Expression of exogenous Irs-2 in Irs-2–/– cells restored the rate of glycolysis to that observed in WT cells. The transcription factor FoxO1 does not appear to be involved in Irs-2-mediated glycolysis. However, Irs-2 does regulate the surface expression of glucose transporter 1 (Glut1) as assessed by flow cytometry using a Glut1-specific ligand. Suppression of Glut1 expression inhibits Irs-2-dependent invasion, which links glycolysis to mammary tumor progression. Irs-2 was shown to be important for mammalian target of rapamycin (mTor) activation, and Irs-2-dependent regulation of Glut1 surface expression is rapamycin-sensitive. Collectively, our data indicate that Irs-2, but not Irs-1, promotes invasion by sustaining the aerobic glycolysis of mouse mammary tumor cells and that it does so by regulating the mTor-dependent surface expression of Glut1.

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.

The role of the extracellular matrix (ECM) in the tumor microenvironment is not limited to being a barrier against tumor invasion. The ECM is a reservoir of cell binding proteins and growth factors that affect tumor cell behavior. It is also substantially modified by proteases produced by tumor cells or stroma cells. As a result of the activity of these proteases, cell-cell and cell-ECM interactions are altered, new biologically active ECM molecules are generated, and the bioavailability and activity of many growth factors, growth factor receptors, and cytokines are modified. ECM-degrading proteases also play a critical role in angiogenesis, where they can act as positive as well as negative regulators of endothelial cell proliferation and vascular morphogenesis. This review article summarizes some of the most relevant findings made over the recent years that were discussed at a workshop organized by the Path B Study Section of the National Institutes of Health in October 2002.

This review highlights an emerging function for vascular endothelial growth factor (VEGF) in carcinoma and discusses mechanisms involved in the elaboration of VEGF autocrine loops. Evidence is provided that autocrine VEGF contributes to the two major components of invasive carcinoma: survival and migration. Moreover, the findings discussed support the hypothesis that carcinoma progression selects for cells that depend on VEGF as a survival factor. Furthermore, a related hypothesis, which is developed, is that the function of the 64 integrin, which has been implicated in carcinoma progression, is linked to its ability to regulate VEGF translation and, consequently, autocrine VEGF signaling. The findings reviewed challenge the notion that the function of VEGF in cancer is limited to angiogenesis and suggest that VEGF and VEGF receptor-based therapeutics, in addition to targeting angiogenesis, may also impair tumor cell survival and invasion directly. © 2003 Published by Elsevier Ltd.

Aberrant cell survival and resistance to apoptosis are hallmarks of tumor invasion and progression to metastatic disease [1], but the mechanisms involved are poorly understood. The epithelial-mesenchymal transition (EMT), a process that facilitates progression to invasive cancer, provides a superb model for studying such survival mechanisms. Here, we used a unique spheroid culture system that recapitulates the structure of the colonic epithelium and undergoes an EMT in response to cytokine stimulation to study this problem [2]. Our data reveal that the EMT results in the increased expression of both VEGF and Flt-1, a tyrosine kinase VEGF receptor, and that the survival of these cells depends on a VEGF/Flt-1 autocrine pathway. Perturbation of Flt-1 function by either a blocking antibody or adenoviral expression of soluble Flt-1, which acts in a dominant-negative fashion, caused massive apoptosis only in cells that underwent EMT. This pathway was critical for the survival of other invasive colon carcinoma cell lines, and we observed a correlative upregulation of Flt-1 expression linked to in vivo human cancer progression. A role for Flt-1 in cell survival is unprecedented and has significant implications for Flt-1 function in tumor progression, as well as in other biological processes, including angiogenesis and development.

The ADAMs (a disintegrin and metalloprotease) comprise a large family of multidomain proteins with cell-binding and metalloprotease activities. The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell attachment using syndecan-4 as a primary cell surface receptor that subsequently triggers beta(1) integrin-dependent cell spreading, stress fiber assembly, and focal adhesion formation. This process contrasts with cell adhesion on fibronectin, which is integrin-initiated but syndecan-4-dependent. In the present study, we investigated ADAM12/syndecan-4 signaling leading to cell spreading and stress fiber formation. We demonstrate that syndecan-4, when present in significant amounts, promotes beta(1) integrin-dependent cell spreading and stress fiber formation in response to rADAM12-cys. A mutant form of syndecan-4 deficient in protein kinase C (PKC)alpha activation or a different member of the syndecan family, syndecan-2, was unable to promote cell spreading. GF109203X and Gö6976, inhibitors of PKC, completely inhibited ADAM12/syndecan-4-induced cell spreading. Expression of syndecan-4, but not syn4DeltaI, resulted in the accumulation of activated beta(1) integrins at the cell periphery in Chinese hamster ovary beta1 cells as revealed by 12G10 staining. Further, expression of myristoylated, constitutively active PKCalpha resulted in beta(1) integrin-dependent cell spreading, but additional activation of RhoA was required to induce stress fiber formation. In summary, these data provide novel insights into syndecan-4 signaling. Syndecan-4 can promote cell spreading in a beta(1) integrin-dependent fashion through PKCalpha and RhoA, and PKCalpha and RhoA likely function in separate pathways.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.

We used published peptide library data to identify PDZ recognition sequences in integrin α subunit cytoplasmic domains and found that the α6A and α5subunits contain a type I PDZ binding site (TSDA*) (asterisk indicates the stop codon). The α6A cytoplasmic domain was used for screening a two-hybrid library to find interacting proteins. The bulk of the captured cDNAs (60%) coded for TIP-2/GIPC, a cytoplasmic protein with one PDZ domain. The interaction of TIP-2/GIPC with different integrin subunits was tested in two-hybrid and in vitro binding assays. Surprisingly, TIP-2/GIPC bound strongly to the C terminus of both α6A and α6B, although the α6B sequence (ESYS*) is not suggestive of a PDZ binding site because of its polar C-terminal residue. For high affinity interaction with TIP-2/GIPC, at least one of the residues at positions –1 and –3 must be negatively charged. An aliphatic residue at position 0 increases the affinity of but is not required for this interaction. The α5 integrin subunit also bound to TIP-2/GIPC. The α6 integrin and TIP-2/GIPC co-localize in retraction fibers in carcinoma cells plated on laminin, a finding suggesting a functional interaction in vivo. Our results demonstrate that both splice variants of α6integrin contain a conserved PDZ binding site that enables interaction with TIP-2/GIPC. The binding site in α6B defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.

Epithelial cells undergo death receptor-dependent apoptosis when detached from matrix, a process termed anoikis. Activation of Akt/protein kinase B (PKB) by matrix attachment protects cells from anoikis. In this study, we establish a link between anoikis and Akt/PKB-mediated survival by demonstrating that Akt/PKB is cleaved by caspases in matrix-detached epithelial cells by a mechanism that involves death receptors. Reduced levels of Akt/PKB protein were observed in detached Madin-Darby canine kidney cells relative to cells attached to collagen. Equivalent levels of Akt/PKB, however, were detected in matrix-adherent and detached cells after inhibition of caspase activity or expression of an Akt/PKB mutant (D108+119A) that is resistant to caspase cleavage. The contribution of death domain-containing proteins to Akt/PKB cleavage was evidenced by the ability of dominant negative Fas-associated death domain to restore normal levels of Akt/PKB in matrix-detached cells. Importantly, expression of a cleavage-resistant Akt/PKB mutant protected matrix-detached cells from apoptosis. These studies suggest that members of the death receptor family promote the caspase-mediated cleavage of Akt/PKB and that this event contributes to anoikis.

This article explores the mechanistic basis of carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression because of their ability to mediate physical interactions with extracellular matrices and their ability to regulate signaling pathways that control actin dynamics and cell movement, and for growth and survival. This article centers on a6 integrins (a6B1 and a6B4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease.

We previously demonstrated that beta(4) integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large beta(4) cytoplasmic domain that are involved in the interaction of alpha(6)beta(4) with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of beta(4) that lacked either all or portions of the beta(4) cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of beta(4) was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of alpha(6)beta(4) to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the beta(4) cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of alpha(6)beta(4) integrin to increase invasion. Interestingly, the extracellular domain of beta(4) is not necessary for alpha(6)beta(4) to stimulate invasion. The association of alpha(6)beta(4) with ErbB-2 is dependent upon the beta(4) cytoplasmic domain and can occur in the absence of alpha(6)beta(4) heterodimerization. Finally, we observed strong activation of PI3K with beta(4) wild type and with those beta(4) deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between alpha(6)beta(4) and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the beta(4) cytoplasmic domain (amino acids 854-1183) in this event.

Background & Aims: Hepatocellular carcinoma is a common complication in liver cirrhosis. The integrin α6β1, a receptor for the laminin family of extracellular matrix proteins, has been found to be overexpressed in hepatocarcinoma. In an effort to further characterize the involvement of α6β1-integrin in hepatocarcinoma progression and to study α6β1-mediated functions, a human hepatocarcinoma cell line, HepG2, that express high surface levels of α6β1 and uses only this integrin to mediate adhesion on laminin was identified. Methods: To assess the role of α6β1 in these cells, a cytoplasmic domain deletion mutant of the β4-integrin subunit by complementary DNA transfection was expressed. The expression of the mutant β4 subunit in association with endogenous α6 showed a dominant-negative effect on α6β1 expression. Results: Stable transfectants of HepG2 that expressed the mutant β4 subunit showed a reduced ability to adhere and migrate on laminin matrices and to invade Matrigel. Furthermore, transfected cells showed significantly lower growth rates and reduced anchorage-independent growth compared with mock-transfected cells. Conclusions: These findings on the expression and function of α6β1 in hepatocarcinoma cells emphasize the potential contribution of this laminin receptor in the neoplastic transformation of hepatocytes. GASTROENTEROLOGY 1998;115:433-442

We examined the possibility that the alpha6A and alpha6B cytoplasmic domain variants of the alpha6beta1 integrin differentially activate p42 and p44 mitogen-activated protein (MAP) kinases. P388D1 macrophages that express equivalent surface levels of either the alpha6Abeta1 or alpha6Bbeta1 integrin were used to examine this issue. Adhesion to laminin-1 mediated by the alpha6Abeta1 integrin triggered activation of a substantial fraction of total p42 and p44 MAP kinases as assessed using a mobility shift assay, immunoblot analysis with a phosphospecific MAP kinase antibody, and an immune complex kinase assay. In contrast, ligation of the alpha6Bbeta1 integrin did not trigger significant MAP kinase activation. These data were confirmed by antibody clustering of the alpha6beta1 integrins. Both the alpha6Abeta1 and alpha6Bbeta1 integrins were capable of activating the p70 ribosomal S6 kinase and this activation, unlike MAP kinase activation, is dependent on phosphoinositide 3-OH kinase. Activation of MAP kinase by alpha6beta1 requires both Ras and protein kinase C activity. A functional correlate for differential activation of MAP kinase was provided by the findings that the alpha6Abeta1 transfectants migrated significantly better on laminin than the alpha6Bbeta1 transfectants and this migration was dependent on MAP kinase activity based on the use of the MAP kinase kinase (MEK1) inhibitor PD98059. Our findings demonstrate that the alpha6beta1 integrin can activate MAP kinase, that this activation is regulated by the cytoplasmic domain of the alpha6 subunit, and that it relates to alpha6beta1-mediated migration.

We demonstrate that the alpha6beta4 integrin promotes carcinoma invasion through a preferential and localized targeting of phosphoinositide-3 OH kinase (PI3K) activity. Stable expression of alpha6beta4 increased carcinoma invasion in a PI3K-dependent manner, and transient expression of a constitutively active PI3K increased invasion in the absence of alpha6beta4. Ligation of alpha6beta4 stimulated significantly more PI3K activity than ligation of beta1 integrins, establishing specificity among integrins for PI3K activation. Alpha6beta4-regulated PI3K activity was required for the formation of lamellae, dynamic sites of motility, in carcinoma cells. The small G protein Rac is required downstream of PI3K for invasion. These studies define a mechanism by which the alpha6beta4 integrin promotes carcinoma invasion and invoke a novel function for PI3K signaling.

Integrin receptors can mediate transmembrane signaling in response to ligand binding. To further examine the role of the integrin α subunit in these signaling functions, we assessed the contribution of the α6 cytoplasmic domain variants to the signaling properties of the α6β1 integrin using P388D1 cells that had been transfected with either the α6A or the α6B cDNA. The α6Aβ1 and α6Bβ1 receptors induced marked quantitative differences in the tyrosine phosphorylation of several proteins after binding to laminin. Specifically, the α6A cytoplasmic domain was more effective than the α6B cytoplasmic domain in inducing the tyrosine phosphorylation of three major proteins (molecular mass, 120, 110, and 76 kDa). In addition to these proteins, we also observed that the tyrosine phosphorylation of the cytoskeletal protein paxillin was increased significantly more by α6Aβ1 integrin-mediated adhesion to laminin than by that of α6Bβ1. This differential pattern of tyrosine phosphorylation induction does not appear to be a secondary event initiated by cell shape changes. Also, differences in tyrosine phosphorylation in the α6 transfectants were not evident in response to attachment to other substrates. These findings provide biochemical evidence for functional differences between α subunit cytoplasmic domain variants of the same integrin.

The integrin alpha 6 beta 4, a receptor for members of the laminin family of basement membrane components, contributes to the function of epithelial cells and their oncogenically transformed derivatives. In our efforts to study alpha 6 beta 4-mediated functions in more detail and to assess the contribution of the beta 4 cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the beta 4 integrin subunit. This cell line, termed RKO, expresses alpha 6 beta 1 but not alpha 6 beta 4, and it interacts with laminin-1 less avidly than similar cell lines that express alpha 6 beta 4. We expressed a full-length beta 4 cDNA, as well as a mutant cDNA that lacks the beta 4 cytoplasmic domain, in RKO cells and isolated stable subclones of these transfectants. In this study, we report that subclones that expressed the full-length beta 4 cDNA in association with endogenous alpha 6 exhibited partial G1 arrest and apoptosis, properties that were not evident in RKO cells transfected with either the cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the alpha 6 beta 4 integrin induced expression of the p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases. These data suggest that the beta 4 integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the beta 4 transfected RKO cells.

Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding.

Rapid progress has been made in understanding the biology of adenocarcinoma of the colon and rectum. Tumor-suppressing genes, mutations in proto-oncogenes, and a gene that increases genetic instability that are associated with the neoplastic transformation of sporadic and familial colorectal carcinomas have been identified. Advances in understanding the genetic alterations that underlie neoplastic transformation suggest that there are at least two distinct pathways for the induction of invasive colorectal carcinoma. Elucidation of both the genetic and phenotypic alterations is slowly leading to better methods of prognostication for patients with carcinoma of the colon or rectum. It soon may be possible to identify and treat individuals at risk for colorectal carcinoma before they develop preneoplastic lesions as well as to target isolated, defined genetic defects for therapy.

Leukocytes use the alpha 6 beta 1 integrin to adhere to laminin based on mAb inhibition and affinity chromatography studies. This adhesion requires leukocyte stimulation with either PMA or specific cytokines, a process that has been termed "inside-out" integrin signaling. In the present study, the involvement of alpha 6 integrin structural variants in this regulated adhesion was examined using mouse macrophages. The two known alpha 6 structural variants, alpha 6A and alpha 6B, differ only in their cytoplasmic domain sequences. Using reverse transcriptase-polymerase chain reaction, we observed that macrophages express only the alpha 6A structural variant, in contrast to most cell types which express both alpha 6A and alpha 6B variants. The role of this integrin subunit in macrophage adhesion was assessed by cDNA transfection of P388D1 cells. We found that this mouse macrophage cell line does not adhere to laminin even in response to phorbol 12-myristate 13-acetate (PMA) stimulation, though it does adhere normally to fibronectin and tissue culture plastic. Subsequent analysis employing reverse transcriptase-polymerase chain reaction and immunoprecipitation of surface labeled cells revealed that this cell line expresses neither the alpha 6A nor alpha 6B integrin subunits. Stable transfection of either the chick or human alpha 6A cDNAs into P388D1 cells resulted in chimeric alpha 6A beta 1 surface expression. The alpha 6A transfectants exhibited inside-out integrin signaling because PMA stimulation markedly increased their ability to adhere to laminin but it did not increase alpha 6A beta 1 surface expression. Similar results were obtained after transfection of the human alpha 6B cDNA. Analysis of the human transfectants was facilitated by the generation of a monoclonal antibody, 2B7, that is specific for the human alpha 6 integrin subunit. These observations demonstrate that both alpha 6A beta 1 and alpha 6B beta 1 can be regulated by inside-out signaling pathways in macrophages, even though this cell type expresses only alpha 6A beta 1. The data presented also demonstrate clearly that the alpha 6A and alpha 6B cytoplasmic domains do not differ in their ability to be regulated by PMA.

Sucrase-isomaltase (SI) is a mucosal disaccharidase that is present in normal small intestine and fetal colon. It also has been noted in colonic adenomas and adenocarcinomas. We used a polyclonal antibody to human SI to investigate enzyme presence and utility in detecting dysplastic changes in chronic ulcerative colitis. Sections from 32 cases were reviewed for the presence or absence of active colitis and dysplasia. Immunostaining of these cases for SI was performed and the results were reported based on location of immunoreactivity (ie, membrane and cytoplasmic staining in superficial and crypt epithelial cells) and percentage of positivity. Of 81 sections examined, 48 were rated negative for dysplasia (23 inactive colitis, 20 active, and five probably negative) and 28 were rated positive (eight low grade and 20 high grade). Surface membrane staining of epithelial cells was noted in all 28 dysplastic slides and positive cases (sensitivity, 100%) but also in 29 of 48 negative sections (P less than .001). In contrast, cytoplasmic positivity was present in 25 of 28 dysplastic and in only two of 48 negative slides (P less than .0001). The presence of cytoplasmic staining of SI in the superficial or crypt cells revealed a sensitivity of 92% and a specificity of 94%. There were five additional sections rated as indefinite for dysplasia (probably positive or unknown); two showed staining patterns typical of negative slides and three showed positive staining patterns. Of the 18 samples of transitional mucosa next to areas of dysplasia, surface membrane staining of SI was seen in all samples and cytoplasmic staining was seen in 15. We conclude that membrane staining of SI can be detected in inflammatory, regenerative, and dysplastic mucosa in ulcerative colitis. Cytoplasmic staining, however, correlates strongly with the presence of dysplastic change and may help in its detection.

Carbohydrate-binding protein 35 (CBP35), also known as the macrophage surface antigen Mac-2, is a lactosamine-specific lectin whose extracellular properties include the ability to agglutinate cells and to bind avidly to the basement membrane glycoprotein laminin. Although these and other properties would be facilitated by dimerization of this lectin, previous studies have argued against multimeric forms of this protein. We report here that macrophage CBP35, purified by laminin affinity chromatography, exists as several distinct species (Mr 35,000, 67,000, and 80,000) when analyzed under non-reducing conditions. This unexpected finding prompted us to study the biochemistry of multimerization using recombinant CBP35 (rCBP35). rCBP35 expressed in Escherichia coli forms disulfide-linked homodimers (Mr 67,000). The dimeric form of CBP35 binds to laminin with higher affinity than does monomer and by a lactosamine-dependent mechanism. Site-directed mutagenesis indicated that cysteine 186, the single cysteine residue in CBP35, is required for dimerization. These results raise the possibility that homo- and heterodimeric forms of CBP35 contribute to its postulated functions in cell-matrix interactions and growth regulation.

Adenocarcinoma of the colon is one of the most prevalent and lethal of all human malignancies. The early diagnosis and management of this disease could be improved if biological markers, whose expression was restricted to malignant colon cells, were identified. Sucrase-isomaltase is a glycoprotein hydrolase expressed throughout the small intestine and fetal colon but not in the normal adult colon. This study shows that the expression of enzymatically active sucrase-isomaltase is a ubiquitous property of primary and metastatic colon adenocarcinoma. Significant sucrase enzyme activity (i.e., > 5 mU/mg protein) was observed in 16 colon carcinomas but not in adjacent normal colon mucosa. Sucrase-isomaltase messenger RNA was identified in all tumors using reverse transcriptase polymerase chain reaction. Using a quantitative polymerase chain reaction analysis, this study shows that the amount of sucrase-isomaltase messenger RNA in tumors examined (3.4 × 10 −8 to 3.19 × 10 −7 μg/μg total RNA) was greater than in adjacent mucosa (0 to 3.4 × 10 −8 μg/μg total RNA). This induction of sucrase-isomaltase messenger RNA and enzyme activity was corroborated by immunostaining. Of 30 colon adenocarcinomas examined, 80% were positive for sucrase-isomaltase. In addition, all colon carcinoma metastases examined were positive for sucrase-isomaltase. The staining pattern was distinct and demarcated tumor cells from the surrounding histologically normal tissue. No sucrase-isomaltase staining was seen in normal mucosa from the same patients. With the exception of lung, no sucrase-isomaltase immunostaining was observed in a variety of examined noncolonic adenocarcinomas. Thus, the specificity and ubiquity of sucrase-isomaltase expression in adenocarcinomas of the colon can be exploited to improve the clinical management of this disease. In addition, studies on the structure of the sucrase-isomaltase gene and its regulatory elements should contribute toward understanding the alteration of gene expression by oncogenic transformation of the colonic mucosa.

Current data indicate that cell adhesion to laminin, the major basement membrane glycoprotein, is mediated by specific integrins, a family of adhesion receptors. In addition, most cell types express a complement of high affinity non-integrin laminin binding proteins (LBPs). Despite considerable effort, the function of these LBPs has not been elucidated. We report here that the major non-integrin LBP of murine macrophages exhibits an Mr of 35,000 and is expressed on the cell surface. Protein microsequencing data revealed that this protein is identical to carbohydrate binding protein 35. This murine galactose-specific lectin is the macrophage antigen Mac-2. Thus, these data suggest that the non-integrin LBPs may contribute to laminin adhesion by a mechanism involving protein-carbohydrate interactions.