Perceptions of Surgical Drains among Breast Reconstruction Patients and Health Care Staff: A Qualitative Survey Study.

BACKGROUND: Surgical drains are a key component for recovery in breast reconstruction procedures. However, they are often cumbersome and carry a risk of infection with prolonged use. We aimed to develop a more thorough understanding of patient and health care provider perspectives on surgical drains, to inform future efforts in improving the breast reconstruction patient experience. METHODS: Twenty-nine breast reconstruction patients and eight plastic surgery providers were recruited to complete surveys focused on surgical drains. Likert scales ranging from 1 to 5 were developed to gauge how bothersome drains felt, as well as concern for infection. Ordinal variable and categorical multiple-choice analyses were applied as appropriate. RESULTS: Fifteen (51.7%) patients underwent implant-based breast reconstruction, and 14 (48.3%) patients underwent autologous breast reconstruction. The most common duration of drain placement was 2 weeks (N = 13). The surgical site infection (SSI) rate requiring antibiotics was 28% (N = 8). On a scale of 1 to 5, both patients (median = 3) and providers (median = 2.5) viewed drains as bothersome. Patients were "frequently" concerned about infection risk (median = 3). Other high-frequency patient concerns included general pain and discomfort. CONCLUSION: Surgical drains are a common component of breast reconstruction procedures and are viewed as cumbersome by both patients and providers. Patients expressed concerns about drain site pain, discomfort, and tugging on clothing. Patients and providers both believed that drains could contribute to SSI. Overall, these data provide insight to drive future improvements in the patient drain experience.

Suppression of Fibrinolysis and Hypercoagulability, Severity of Hypoxemia, and Mortality in COVID-19 Patients: A Retrospective Cohort Study.

BACKGROUND: COVID-19 causes hypercoagulability, but the association between coagulopathy and hypoxemia in critically ill patients has not been thoroughly explored. This study hypothesized that severity of coagulopathy would be associated with acute respiratory distress syndrome severity, major thrombotic events, and mortality in patients requiring intensive care unit-level care. METHODS: Viscoelastic testing by rotational thromboelastometry and coagulation factor biomarker analyses were performed in this prospective observational cohort study of critically ill COVID-19 patients from April 2020 to October 2020. Statistical analyses were performed to identify significant coagulopathic biomarkers such as fibrinolysis-inhibiting plasminogen activator inhibitor 1 and their associations with clinical outcomes such as mortality, extracorporeal membrane oxygenation requirement, occurrence of major thrombotic events, and severity of hypoxemia (arterial partial pressure of oxygen/fraction of inspired oxygen categorized into mild, moderate, and severe per the Berlin criteria). RESULTS: In total, 53 of 55 (96%) of the cohort required mechanical ventilation and 9 of 55 (16%) required extracorporeal membrane oxygenation. Extracorporeal membrane oxygenation-naïve patients demonstrated lysis indices at 30 min indicative of fibrinolytic suppression on rotational thromboelastometry. Survivors demonstrated fewer procoagulate acute phase reactants, such as microparticle-bound tissue factor levels (odds ratio, 0.14 [0.02, 0.99]; P = 0.049). Those who did not experience significant bleeding events had smaller changes in ADAMTS13 levels compared to those who did (odds ratio, 0.05 [0, 0.7]; P = 0.026). Elevations in plasminogen activator inhibitor 1 (odds ratio, 1.95 [1.21, 3.14]; P = 0.006), d-dimer (odds ratio, 3.52 [0.99, 12.48]; P = 0.05), and factor VIII (no clot, 1.15 ± 0.28 vs. clot, 1.42 ± 0.31; P = 0.003) were also demonstrated in extracorporeal membrane oxygenation-naïve patients who experienced major thrombotic events. Plasminogen activator inhibitor 1 levels were significantly elevated during periods of severe compared to mild and moderate acute respiratory distress syndrome (severe, 44.2 ± 14.9 ng/ml vs. mild, 31.8 ± 14.7 ng/ml and moderate, 33.1 ± 15.9 ng/ml; P = 0.029 and 0.039, respectively). CONCLUSIONS: Increased inflammatory and procoagulant markers such as plasminogen activator inhibitor 1, microparticle-bound tissue factor, and von Willebrand factor levels are associated with severe hypoxemia and major thrombotic events, implicating fibrinolytic suppression in the microcirculatory system and subsequent micro- and macrovascular thrombosis in severe COVID-19.

The Association Between Metabolic Derangement and Wound Complications in Elective Plastic Surgery.

INTRODUCTION: The incidence of metabolically unhealthy obesity is rising nationally. In this study, we compare wound and overall complications between metabolically unhealthy obese and healthy patients undergoing elective plastic surgery and model how operative time influences a complication risk. METHODS: Patients undergoing elective breast and body plastic surgery procedures in the 2009-2019 National Surgical Quality Improvement Program (NSQIP) dataset were identified. Complications were compared between metabolically unhealthy obese (body mass index [BMI] > 30 with diabetes and/or hypertension) versus metabolically healthy obese patients (BMI > 30 without diabetes or hypertension). Logistic regression was used to model the probability of wound complications across operative times stratified by metabolic status. RESULTS: Of 139,352 patients, 13.4% (n = 18,663) had metabolically unhealthy obesity and 23.8% (n = 33,135) had metabolically healthy obesity. Compared to metabolically healthy patients, metabolically unhealthy patients had higher incidence of wound complications (6.9% versus 5.6%; P < 0.001) and adverse events (12.4% versus 9.6%; P < 0.001), in addition to higher 30-d readmission, returns to the operating room, and length of stay (all P < 0.001). After adjustment, BMI (Odds ratio [OR] 7.86), hypertension (OR 1.15), and diabetes (OR 1.25) were independent risk factors for wound complications (all P < 0.001). Among metabolically unhealthy patients, the operative time was log-linear with a wound complication risk (OR 1.21; P < 0.001). CONCLUSIONS: Diabetes and hypertension are additive risk factors with obesity for wound complications in elective plastic surgery. Among patients with metabolically unhealthy obesity, a risk of wound complications increases logarithmically with operative time. This distinction with regard to metabolic state might explain the unclear impact of obesity on surgical outcomes within existing surgical literature.

Adipose stem cell crosstalk with chemo-residual breast cancer cells: implications for tumor recurrence.

PURPOSE: Most triple-negative breast cancer (TNBC) patients exhibit an incomplete response to neoadjuvant chemotherapy, resulting in chemo-residual tumor cells that drive tumor recurrence and patient mortality. Accordingly, strategies for eliminating chemo-residual tumor cells are urgently needed. Although stromal cells contribute to tumor cell invasion, to date, their ability to influence chemo-residual tumor cell behavior has not been examined. Our study is the first to investigate cross-talk between adipose-derived stem cells (ASCs) and chemo-residual TNBC cells. We examine if ASCs promote chemo-residual tumor cell proliferation, having implications for tumor recurrence. METHODS: ASC migration toward chemo-residual TNBC cells was tested in a transwell migration assay. Importance of the SDF-1α/CXCR4 axis was determined using neutralizing antibodies and a small molecule inhibitor. The ability of ASCs to drive tumor cell proliferation was analyzed by culturing tumor cells ± ASC conditioned media (CM) and determining cell counts. Downstream signaling pathways activated in chemo-residual tumor cells following their exposure to ASC CM were studied by immunoblotting. Importance of FGF2 in promoting proliferation was assessed using an FGF2-neutralizing antibody. RESULTS: ASCs migrated toward chemo-residual TNBC cells in a CXCR4/SDF-1α-dependent manner. Moreover, ASC CM increased chemo-residual tumor cell proliferation and activity of extracellular signal-regulated kinase (ERK). An FGF2-neutralizing antibody inhibited ASC-induced chemo-residual tumor cell proliferation. CONCLUSIONS: ASCs migrate toward chemo-residual TNBC cells via SDF-1α/CXCR4 signaling, and drive chemo-residual tumor cell proliferation in a paracrine manner by secreting FGF2 and activating ERK. This paracrine signaling can potentially be targeted to prevent tumor recurrence.

Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

INTRODUCTION: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)]. METHODS: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy. RESULTS: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells. CONCLUSION: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

Cancer Mutations Converge on a Collection of Protein Assemblies to Predict Resistance to Replication Stress.

Rapid proliferation is a hallmark of cancer associated with sensitivity to therapeutics that cause DNA replication stress (RS). Many tumors exhibit drug resistance, however, via molecular pathways that are incompletely understood. Here, we develop an ensemble of predictive models that elucidate how cancer mutations impact the response to common RS-inducing (RSi) agents. The models implement recent advances in deep learning to facilitate multidrug prediction and mechanistic interpretation. Initial studies in tumor cells identify 41 molecular assemblies that integrate alterations in hundreds of genes for accurate drug response prediction. These cover roles in transcription, repair, cell-cycle checkpoints, and growth signaling, of which 30 are shown by loss-of-function genetic screens to regulate drug sensitivity or replication restart. The model translates to cisplatin-treated cervical cancer patients, highlighting an RTK-JAK-STAT assembly governing resistance. This study defines a compendium of mechanisms by which mutations affect therapeutic responses, with implications for precision medicine. SIGNIFICANCE: Zhao and colleagues use recent advances in machine learning to study the effects of tumor mutations on the response to common therapeutics that cause RS. The resulting predictive models integrate numerous genetic alterations distributed across a constellation of molecular assemblies, facilitating a quantitative and interpretable assessment of drug response. This article is featured in Selected Articles from This Issue, p. 384.

Evaluation of large language models for discovery of gene set function.

Gene set analysis is a mainstay of functional genomics, but it relies on curated databases of gene functions that are incomplete. Here we evaluate five Large Language Models (LLMs) for their ability to discover the common biological functions represented by a gene set, substantiated by supporting rationale, citations and a confidence assessment. Benchmarking against canonical gene sets from the Gene Ontology, GPT-4 confidently recovered the curated name or a more general concept (73% of cases), while benchmarking against random gene sets correctly yielded zero confidence. Gemini-Pro and Mixtral-Instruct showed ability in naming but were falsely confident for random sets, whereas Llama2-70b had poor performance overall. In gene sets derived from 'omics data, GPT-4 identified novel functions not reported by classical functional enrichment (32% of cases), which independent review indicated were largely verifiable and not hallucinations. The ability to rapidly synthesize common gene functions positions LLMs as valuable 'omics assistants.

A deep learning model of tumor cell architecture elucidates response and resistance to CDK4/6 inhibitors.

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6is) have revolutionized breast cancer therapy. However, <50% of patients have an objective response, and nearly all patients develop resistance during therapy. To elucidate the underlying mechanisms, we constructed an interpretable deep learning model of the response to palbociclib, a CDK4/6i, based on a reference map of multiprotein assemblies in cancer. The model identifies eight core assemblies that integrate rare and common alterations across 90 genes to stratify palbociclib-sensitive versus palbociclib-resistant cell lines. Predictions translate to patients and patient-derived xenografts, whereas single-gene biomarkers do not. Most predictive assemblies can be shown by CRISPR-Cas9 genetic disruption to regulate the CDK4/6i response. Validated assemblies relate to cell-cycle control, growth factor signaling and a histone regulatory complex that we show promotes S-phase entry through the activation of the histone modifiers KAT6A and TBL1XR1 and the transcription factor RUNX1. This study enables an integrated assessment of how a tumor's genetic profile modulates CDK4/6i resistance.

Evaluation of large language models for discovery of gene set function.

Gene set analysis is a mainstay of functional genomics, but it relies on manually curated databases of gene functions that are incomplete and unaware of biological context. Here we evaluate the ability of OpenAI's GPT-4, a Large Language Model (LLM), to develop hypotheses about common gene functions from its embedded biomedical knowledge. We created a GPT-4 pipeline to label gene sets with names that summarize their consensus functions, substantiated by analysis text and citations. Benchmarking against named gene sets in the Gene Ontology, GPT-4 generated very similar names in 50% of cases, while in most remaining cases it recovered the name of a more general concept. In gene sets discovered in 'omics data, GPT-4 names were more informative than gene set enrichment, with supporting statements and citations that largely verified in human review. The ability to rapidly synthesize common gene functions positions LLMs as valuable functional genomics assistants.

Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells.

Translation of the small G protein RhoA in neurons is regulated by the eukaryotic translation initiation factor eIF4E. Here we show that this translation factor also regulates RhoA expression and activity in breast cancer cells. The introduction of eIF4E into breast tumor cells increased RhoA protein levels, while expression of an eIF4E siRNA reduced RhoA expression. Previous studies indicate that the axon repulsion factor Semaphorin3A (Sema3A) stimulates the eIF4E-dependent translation of RhoA in neurons, and breast tumor cells support autocrine Sema3A signaling. Accordingly, we next examined if autocrine Sema3A signaling drives eIF4E-dependent RhoA translation in breast cancer cells. The incubation of breast tumor cells with recombinant Sema3A rapidly increased eIF4E activity, RhoA protein levels, and RhoA activity. This Sema3A activity was blocked in tumor cells expressing an shRNA-specific for the Sema3A receptor, Neuropilin-1 (NP-1), as well as in cells incubated with an eIF4E inhibitor. Importantly, RhoA protein levels were reduced in Sema3A shRNA-expressing compared to control shRNA-expressing breast tumor cells, demonstrating that autocrine Sema3A increases RhoA expression in breast cancer. Considering that Sema3A suppresses axon extension by stimulating RhoA translation, we next examined if the Sema3A/RhoA axis impacts breast tumor cell migration. The incubation of control breast tumor cells, but not RhoA shRNA-expressing cells, with rSema3A significantly reduced their migration. Collectively, these studies indicate that Sema3A impedes breast tumor cell migration in part by stimulating RhoA. These findings identify common signaling pathways that regulate the navigation of neurons and breast cancer cells, thus suggesting novel targets for suppressing breast tumor cell migration.

Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition.

We report that the activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial-mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. We identify NFkappaB as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition.

Autocrine semaphorin3A stimulates alpha2 beta1 integrin expression/function in breast tumor cells.

The axon repulsion factor semaphorin3A (SEMA3A) and its receptor neuropilin-1 (NP-1) are expressed in breast tumor cells, and function as suppressors of tumor cell migration. Based on the knowledge that both SEMA3A and the alpha2beta1 integrin suppress breast tumor cell migration, we studied the impact of SEMA3A signaling on alpha2beta1 integrin expression/function. The incubation of breast tumor cells with SEMA3A increased alpha2 and beta1 integrin levels, and stimulated tumor cell adhesion to the alpha2beta1-binding matrix protein collagen I. Conversely, reducing SEMA3A expression in breast tumor cells decreased alpha2beta1 levels and collagen adhesion. The ability of SEMA3A to increase tumor cell adhesion to collagen was dependent on both the SEMA3A receptor NP-1 and the glycogen synthase kinase-3. The incubation of breast tumor cells with SEMA3A disrupted the actin cytoskeleton, and reduced both tumor cell migratory and invasive behavior. Importantly, using an alpha2beta1-neutralizing antibody, we demonstrated that SEMA3A suppression of tumor cell migration is dependent on alpha2beta1. Our studies indicate that expression of the alpha2beta1 integrin, a suppressor of metastatic breast tumor growth, is stimulated in breast tumor cells by an autocrine SEMA3A pathway.

Integrin (alpha 6 beta 4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells.

We define a novel mechanism by which integrins regulate growth factor expression and the survival of carcinoma cells. Specifically, we demonstrate that the alpha 6 beta 4 integrin enhances vascular endothelial growth factor (VEGF) translation in breast carcinoma cells. The mechanism involves the ability of this integrin to stimulate the phosphorylation and inactivation of 4E-binding protein (4E-BP1), a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (eIF-4E). The regulation of 4E-BP1 phosphorylation by alpha 6 beta 4 derives from the ability of this integrin to activate the PI-3K-Akt pathway and, consequently, the rapamycin-sensitive kinase mTOR that can phosphorylate 4E-BP1. Importantly, we show that this alpha 6 beta 4-dependent regulation of VEGF translation plays an important role in the survival of metastatic breast carcinoma cells by sustaining a VEGF autocrine signaling pathway that involves activation of PI-3K and Akt. These findings reveal that integrin-mediated activation of PI-3K-Akt is amplified by integrin-stimulated VEGF expression and they provide a mechanism that substantiates the reported role of alpha 6 beta 4 in carcinoma progression.

Vascular endothelial growth factor-A stimulates Snail expression in breast tumor cells: implications for tumor progression.

The E-cadherin transcriptional repressor Snail is a prognostic marker for metastatic breast carcinoma, as well as a critical determinant of tumor growth and recurrence. We define a non-angiogenic, autocrine function for the vascular endothelial growth factor-A (VEGF-A) in regulating Snail expression in breast tumor cells. The transfection of well-differentiated breast tumor cells with VEGF-A increases Snail mRNA and protein levels, resulting in reduced E-cadherin expression. Conversely, reducing endogenous VEGF-A expression in poorly differentiated breast tumor cells by siRNA transfection decreases Snail levels. Our studies demonstrate that VEGF and the VEGF receptor Neuropilin-1 increase Snail expression by suppressing the Glycogen Synthase Kinase-3 (GSK-3), an established inhibitor of Snail transcription and protein stability. The VEGF-A neutralizing antibody Avastin was recently approved by the FDA for the treatment of metastatic breast cancer. We present the provocative finding that beyond its anti-angiogenic activity, Avastin can reduce Snail expression in breast tumor cells. Collectively, this work describes a novel autocrine function for VEGF in breast tumor cells in driving the expression of Snail, a breast tumor progression factor. Based on our demonstration that Avastin reduces Snail expression in breast tumor cells, we propose that the treatment of early stage breast cancer patients with Avastin may impede tumor progression.

The alpha6beta4 integrin can regulate ErbB-3 expression: implications for alpha6beta4 signaling and function.

The integrin alpha(6)beta(4) has been shown to facilitate key functions of carcinoma cells, including their ability to migrate, invade, and evade apoptosis. The mechanism involved seems to be a profound effect of alpha(6)beta(4) on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An intimate relationship between alpha(6)beta(4) and growth factor receptors may explain this effect of alpha(6)beta(4) on signaling. Previously, we showed that alpha(6)beta(4) and ErbB-2 can function synergistically to activate the PI3K/Akt pathway. Given that ErbB-2 can activate PI3K only when it heterodimerizes with other members of the epidermal growth factor receptor family, these data imply that other receptors cooperate in this process. Here, we report that alpha(6)beta(4) can regulate the expression of ErbB-3 using several different models and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the alpha(6)beta(4)-dependent activation of PI3K/Akt and the ability of this integrin to impede apoptosis of carcinoma cells. Our data also support the hypothesis that alpha(6)beta(4) can regulate ErbB-3 expression at the translational level as evidenced by the findings that alpha(6)beta(4) does not increase ErbB-3 mRNA significantly, and that this regulation is both rapamycin sensitive and dependent on eukaryotic translation initiation factor 4E. These findings provide one mechanism to account for the activation of PI3K by alpha(6)beta(4) and they also provide insight into the regulation of ErbB-3 in carcinoma cells.

CRISPR/Cas9-Mediated BRCA1 Knockdown Adipose Stem Cells Promote Breast Cancer Progression.

BACKGROUND: The tumor microenvironment within the breast is rich in adipose elements. The interaction between adipose cells and breast cancer is poorly understood, particularly as it pertains to patients with genetic susceptibility to breast cancer. This study focuses on the phenotype of human adipose-derived stem cells with the BRCA1 mutation and the effect they may have on breast cancer cell behavior. METHODS: CRISPR/Cas9 was used to generate de novo BRCA1-knockdown human adipose-derived stem cells. The effect of the BRCA1 knockdown on the adipose-derived stem cell phenotype was compared to wild-type adipose-derived stem cells and patient-derived breast adipose-derived stem cells with known BRCA1 mutations. Interactions between adipose-derived stem cells and the MDA-MB-231 breast cancer cell line were evaluated. RESULTS: BRCA1-knockdown adipose-derived stem cells stimulated MDA-MB-231 proliferation (1.4-fold increase on day 4; p = 0.0074) and invasion (2.3-fold increase on day 2; p = 0.0171) compared to wild-type cells. Immunofluorescence staining revealed higher levels of phosphorylated ataxia telangiectasia-mutated activation in BRCA1-knockdown cells (72.9 ± 5.32 percent versus 42.9 ± 4.97 percent; p = 0.0147), indicating higher levels of DNA damage. Beta-galactosidase staining demonstrated a significantly higher level of senescence in BRCA1-knockdown cells compared with wild-type cells (7.9 ± 0.25 percent versus 0.17 ± 0.17 percent; p < 0.0001). Using quantitative enzyme-linked immunosorbent assay to evaluate conditioned media, the authors found significantly higher levels of interleukin-8 in BRCA1-knockdown cells (2.57 ± 0.32-fold; p = 0.0049). CONCLUSIONS: The authors show for the first time that the BRCA1 mutation affects the adipose-derived stem cell phenotype. Moreover, CRISPR/Cas9-generated BRCA1-knockdown adipose-derived stem cells stimulate a more aggressive behavior in breast cancer cells than wild-type adipose-derived stem cells. This appears to be related to increased inflammatory cytokine production by means of a DNA damage-mediated cell senescence pathway.

Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids.

Aberrant cell survival and resistance to apoptosis are hallmarks of tumor invasion and progression to metastatic disease, 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. 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.

Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4.

We report that vascular endothelial growth factor (VEGF), a major angiogenic factor, is also arequisite autocrine factor for breast carcinoma invasion in vitro and that the VEGF receptor Neuropilin-1 but not Flt-1 is essential for this function. VEGF regulates expression of the chemokine receptor CXCR4, and this VEGF target is needed for invasion but not for cell survival. CXCR4 mediates migration of breast carcinoma cells toward stromal-derived factor-1, and this migration is dependent on autocrine VEGF. Of interest, a CXCR4-inhibitory peptide that is currently in HIV clinical trials suppressed invasion. Our findings indicate that a VEGF autocrine pathway induces chemokine receptor expression in breast carcinoma cells, thus promoting their directed migration toward specific chemokines.

Hypoxia-induced vascular endothelial growth factor transcription and protection from apoptosis are dependent on alpha6beta1 integrin in breast carcinoma cells.

The alpha6beta1 integrin has been implicated in breast carcinoma progression, but the mechanisms involved remain elusive. MDA-MB-435 cells engineered to be deficient in alpha6beta1 expression form primary tumors that are highly apoptotic and unable to metastasize, although they exhibit no increased apoptosis in vitro under standard culture conditions. Based on the hypothesis that alpha6beta1 is necessary for the survival of these cells in the tumor microenvironment, we report here that hypoxia protects these cells from apoptosis induced by serum deprivation and that hypoxia-mediated protection requires alpha6beta1 expression. We investigated the influence of alpha6beta1 on vascular endothelial growth factor (VEGF) expression because autocrine VEGF is necessary for the survival of serum-deprived cells in hypoxia. The results obtained indicate that alpha6beta1 is necessary for VEGF expression because the ability of hypoxia to activate HIF-1 and to stimulate VEGF transcription in MDA-MB-435 cells is dependent on alpha6beta1 expression by a mechanism that involves protein kinase C-alpha.