Expression of metastasis suppressor BRMS1 in breast cancer cells results in a marked delay in cellular adhesion to matrix.

Metastatic dissemination is a multi-step process that depends on cancer cells' ability to respond to microenvironmental cues by adapting adhesion abilities and undergoing cytoskeletal rearrangement. Breast Cancer Metastasis Suppressor 1 (BRMS1) affects several steps of the metastatic cascade: it decreases survival in circulation, increases susceptibility to anoikis, and reduces capacity to colonize secondary organs. In this report, BRMS1 expression is shown to not significantly alter expression levels of integrin monomers, while time-lapse and confocal microscopy revealed that BRMS1-expressing cells exhibited reduced activation of both ß1 integrin and focal adhesion kinase, and decreased localization of these molecules to sites of focal adhesions. Short-term plating of BRMS1-expressing cells onto collagen or fibronectin markedly decreased cytoskeletal reorganization and formation of cellular adhesion projections. Under 3D culture conditions, BRMS1-expressing cells remained rounded and failed to reorganize their cytoskeleton and form invasive colonies. Taken together, BRMS1-expressing breast cancer cells are greatly attenuated in their ability to respond to microenvironment changes. © 2013 Wiley Periodicals, Inc.

Unraveling the 'TGF-ß paradox' one metastamir at a time.

Transforming growth factor beta (TGF-ß) has received noteworthy attention in the recent past due to its unique characteristic of functionally switching roles from tumor suppressor to metastasis promoter. To uncover the black box surrounding the mechanisms of TGF-ß, Taylor and colleagues performed global miRNA expression analyses using a murine mammary carcinoma progression model. They discovered multiple miRNA regulated by TGF-ß and matrix stiffness. Focusing on miR-181a, they uncovered an intricate pathway regulating breast cancer metastasis that sheds new insight into metastasis regulation that may prove useful in clinical settings.

Metastasis suppression by BRMS1 associated with SIN3 chromatin remodeling complexes.

Epigenetic regulation of gene transcription by histone modification and chromatin remodeling has been linked to many biological and pathological events including cancer metastasis. Breast cancer metastasis suppressor 1 (BRMS1) interacts with SIN3 chromatin remodeling complexes, and, upon forced expression in metastatic cells, a nearly complete suppression of metastasis is noted without preventing primary tumor growth. The data for BRMS1-mediated metastasis suppression and SIN3 interaction are clear; however, connecting the inhibition directly to the association of BRMS1 with SIN3 complexes is currently not well defined. Considering the recent advancements in developing epigenetic drugs for cancer therapy, an improved understanding of how the interactions between BRMS1 and SIN3 regulate the process of metastasis should lead to novel therapies specifically targeting the most deadly aspect of tumor progression. In this article, the data for BRMS1-mediated metastasis suppression are reviewed with a focus on how the SIN3 chromatin remodeling complexes may be functionally involved.

Metastasis suppressors and the tumor microenvironment.

The most lethal and debilitating attribute of cancer cells is their ability to metastasize. Throughout the process of metastasis, tumor cells interact with other tumor cells, host cells and a variety of molecules. Tumor cells are also faced with a number of insults, such as hemodynamic sheer pressure and immune selection. This brief review explores how metastasis suppressor proteins regulate interactions between tumor cells and the microenvironments in which tumor cells find themselves.

Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive tumor phenotype.

Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype.

Clinical significance of KISS1 protein expression for brain invasion and metastasis.

BACKGROUND: Metastases to the brain represent a feared complication and contribute to the morbidity and mortality of breast cancer. Despite improvements in therapy, prognostic factors for development of metastases are lacking. KISS1 is a metastasis suppressor that demonstrates inhibition of metastases formation in several types of cancer. The purpose of this study was to determine the importance of KISS1 expression in breast cancer progression and the development of intracerebral lesions. METHODS: In this study, we performed a comparative analysis of 47 brain metastases and 165 primary breast cancer specimens by using the antihuman KISS1 antibody. To compare KISS1 expression between different groups, we used a 3-tier score and the automated score computer software (ACIS) evaluation. To reveal association between mRNA and protein expression, we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Significance of immunohistochemistry stainings was correlated with clinicopathological data. RESULTS: We identified that KISS1 expression is significantly higher in primary breast cancer compared with brain metastases (P < .05). The mRNA analysis performed on 33 selected ductal carcinoma brain metastatic lesions and 36 primary ductal carcinomas revealed a statistically significant down-regulation of KISS1 protein in metastatic cases (P = .04). Finally, we observed a significant correlation between expression of KISS1 and metastasis-free survival (P = .04) along with progression of breast cancer and expression of KISS1 in primary breast cancer specimens (P = .044). CONCLUSIONS: In conclusion, our study shows that breast cancer expresses KISS1. Cytoplasmic expression of KISS1 may be used as a prognostic marker for increased risk of breast cancer progression.

Suppression of SIN3A by miR-183 Promotes Breast Cancer Metastasis.

Recent work has established that SWI-independent-3 (SIN3) chromatin modification complexes play key roles in cancer progression. We previously demonstrated that knockdown of SIN3A expression promotes human breast cancer cell invasion and metastasis; however, the levels of SIN3A in patient breast carcinoma are not known. We therefore examined SIN3A mRNA and protein in patient tissues and determined that SIN3A expression is lower in breast carcinoma relative to normal breast. Given the 3'-untranslated region (UTR) of SIN3A has several conserved binding sites for oncogenic miRNA, we hypothesized that SIN3A is targeted by miRNA and found that ectopic miR-183 results in decreased SIN3A in breast carcinoma cell lines. Functionally, we demonstrate that miR-183 promotes breast cancer cell migration and invasion in a SIN3A-dependent manner and ectopic miR-183 promotes metastasis in vivo. Patients with breast cancer with high levels of miR-183 and low levels of SIN3A have the shortest overall survival. Given the critical link between metastasis and survival in patients with breast cancer, it is of utmost importance to identify clinically relevant genes involved in metastasis. Here, we report for the first time the aberrant expression of the putative metastasis suppressing gene SIN3A in human breast cancers and propose a mechanism of SIN3A suppression by miR-183. IMPLICATIONS: SIN3A expression is decreased in metastatic breast cancer in part due to miR-183.

Perturbation of BRMS1 interactome reveals pathways that impact metastasis.

Breast Cancer Metastasis Suppressor 1 (BRMS1) expression is associated with longer patient survival in multiple cancer types. Understanding BRMS1 functionality will provide insights into both mechanism of action and will enhance potential therapeutic development. In this study, we confirmed that the C-terminus of BRMS1 is critical for metastasis suppression and hypothesized that critical protein interactions in this region would explain its function. Phosphorylation status at S237 regulates BRMS1 protein interactions related to a variety of biological processes, phenotypes [cell cycle (e.g., CDKN2A), DNA repair (e.g., BRCA1)], and metastasis [(e.g., TCF2 and POLE2)]. Presence of S237 also directly decreased MDA-MB-231 breast carcinoma migration in vitro and metastases in vivo. The results add significantly to our understanding of how BRMS1 interactions with Sin3/HDAC complexes regulate metastasis and expand insights into BRMS1's molecular role, as they demonstrate BRMS1 C-terminus involvement in distinct protein-protein interactions.

miR-31 Displays Subtype Specificity in Lung Cancer.

miRNA rarely possess pan-oncogenic or tumor-suppressive properties. Most miRNAs function under tissue-specific contexts, acting as either tumor suppressors in one tissue, promoting oncogenesis in another, or having no apparent role in the regulation of processes associated with the hallmarks of cancer. What has been less clear is the role of miRNAs within cell types of the same tissue and the ability within each cell type to contribute to oncogenesis. In this study, we characterize the role of one such tissue-specific miRNA, miR-31, recently identified as the most oncogenic miRNA in lung adenocarcinoma, across the histologic spectrum of human lung cancer. Compared with normal lung tissue, miR-31 was overexpressed in patient lung adenocarcinoma, squamous cell carcinoma, and large-cell neuroendocrine carcinoma, but not small-cell carcinoma or carcinoids. miR-31 promoted tumor growth in mice of xenografted human adenocarcinoma and squamous cell carcinoma cell lines, but not in large- or small-cell carcinoma lines. While miR-31 did not promote primary tumor growth of large- and small-cell carcinoma, it did promote spontaneous metastasis. Mechanistically, miR-31 altered distinct cellular signaling programs within each histologic subtype, resulting in distinct phenotypic differences. This is the first report distinguishing diverse functional roles for this miRNA across the spectrum of lung cancers and suggests that miR-31 has broad clinical value in human lung malignancy. SIGNIFICANCE: These findings demonstrate the oncogenic properties of miR-31 in specific subtypes of lung cancer and highlight it as a potential therapeutic target in these subtypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/1942/F1.large.jpg.

Re-expression of DIRAS3 and p53 induces apoptosis and impaired autophagy in head and neck squamous cell carcinoma.

BACKGROUND: p53 and DIRAS3 are tumor suppressors that are frequently silenced in tumors. In this study, we sought to determine whether the concurrent re-expression of p53 and DIRAS3 could effectively induce head and neck squamous cell carcinoma (HNSCC) cell death. METHODS: CAL-27 and SCC-25 cells were treated with Ad-DIRAS3 and rAd-p53 to induce re-expression of DIRAS3 and p53 respectively. The effects of DIRAS3 and p53 re-expression on the growth and apoptosis of HNSCC cells were examined by TUNEL assay, flow cytometric analysis and MTT. The effects of DIRAS3 and p53 re-expression on Akt phosphorylation, oncogene expression, and the interaction of 4E-BP1 with eIF4E were determined by real-time PCR, Western blotting and immunoprecipitation analysis. The ability of DIRAS3 and p53 re-expression to induce autophagy was evaluated by transmission electron microscopy, LC3 fluorescence microscopy and Western blotting. The effects of DIRAS3 and p53 re-expression on HNSCC growth were evaluated by using an orthotopic xenograft mouse model. RESULTS: TUNEL assay and flow cytometric analysis showed that the concurrent re-expression of DIRAS3 and p53 significantly induced apoptosis (P < 0.001). MTT and flow cytometric analysis revealed that DIRAS3 and p53 re-expression significantly inhibited proliferation and induced cell cycle arrest (P < 0.001). Mechanistically, the concurrent re-expression of DIRAS3 and p53 down-regulated signal transducer and activation of transcription 3 (STAT3) and up-regulated p21WAF1/CIP1 and Bax (P < 0.001). DIRAS3 and p53 re-expression also inhibited Akt phosphorylation, increased the interaction of eIF4E with 4E-BP1, and reduced the expression of c-Myc, cyclin D1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor receptor (EGFR) and Bcl-2 (P < 0.001). Moreover, the concurrent re-expression of DIRAS3 and p53 increased the percentage of cells with GFP-LC3 puncta compared with that in cells treated with control adenovirus (50.00% ± 4.55% vs. 4.67% ± 1.25%, P < 0.001). LC3 fluorescence microscopy and Western blotting further showed that DIRAS3 and p53 re-expression significantly promoted autophagic activity but also inhibited autophagic flux, resulting in overall impaired autophagy. Finally, the concurrent re-expression of DIRAS3 and p53 significantly decreased the tumor volume compared with the control group in a HNSCC xenograft mouse model [(3.12 ± 0.75) mm3 vs. (189.02 ± 17.54) mm3, P < 0.001]. CONCLUSIONS: The concurrent re-expression of DIRAS3 and p53 is a more effective approach to HNSCC treatment than current treatment strategies.

Long Noncoding RNA HITTERS Protects Oral Squamous Cell Carcinoma Cells from Endoplasmic Reticulum Stress-Induced Apoptosis via Promoting MRE11-RAD50-NBS1 Complex Formation.

Recent studies have proven that long noncoding RNAs (lncRNAs) exhibit regulatory functions of both DNA damage response (DDR) and endoplasmic reticulum (ER) stress. Herein, ER stress-induced lncRNA transcriptomic changes are reported in human oral squamous cell carcinoma (OSCC) cells and a novel lncRNA HITTERS ( H ERPUD1 intronic transcript of ER stress) is identified as the most significantly upregulated lncRNA. It is shown that HITTERS is a nucleus-located lncRNA including two transcript variants. HITTERS lacks an independent promoter but shares the same promoter with HERPUD1. HITTERS is transcriptionally regulated by Activating Transcription Factor (ATF) 6, ATF4, X-Box Binding Protein 1 (XBP1), and DNA methylation. In human OSCC tissues, HITTERS is significantly correlated with OSCC clinicopathological features and prognosis. Gain- and loss-of-function studies reveal that HITTERS promotes OSCC proliferation and invasion via influencing the expression of growth factor receptors and the downstream pathways. Once ER stress is triggered, HITTERS significantly attenuates ER stress-induced apoptosis both in vivo and in vitro. Mechanically, HITTERS functions as RNA scaffold to promote MRE11-RAD50-NBS1 complex formation in the repair of ER stress-induced DNA damage. To sum up, this study presents a novel lncRNA, namely HITTERS, which links ER stress and DDR together in OSCC.

Breast cancer metastasis suppressor 1 coordinately regulates metastasis-associated microRNA expression.

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis-associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. MicroRNA arrays were done comparing small RNAs that were purified from metastatic MDA-MB-231 and MDA-MB-435 and their nonmetastatic BRMS1-transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT-PCR. BRMS1 decreased metastasis-promoting (miR-10b, -373 and -520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR-10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR-146a, -146b and -335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasis-associated miRNA and suggests that recruitment of BRMS1-containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis.

Inhibition of CXCR4 by CTCE-9908 inhibits breast cancer metastasis to lung and bone.

Metastasis occurs, in part, due to tumor cell responses to chemokine secretion by ectopic organs or tissues. SDF-1 is constitutively expressed in tissues where metastases frequently develop while breast carcinoma cells express the receptor for SDF-1, CXCR4, which is correlated with increased bone metastasis and poor overall survival. We hypothesized that treatment with a CXCR4 antagonist, CTCE-9908, would decrease incidence of bone and lung metastasis. Treatment with CTCE-9908 (25 mg/kg) began the day prior to or the day of intravenous or intracardiac tumor cell inoculation of MDA-MB-231 human breast carcinoma cells expressing enhanced green fluorescent protein (GFP) into athymic mice. After 5 or 8 weeks (i.c. and i.v. injections, respectively), the presence of fluorescent foci at metastatic sites was assessed. Somewhat surprisingly, CTCE-9908 treatment did not decrease incidence of metastasis as hypothesized. However, CTCE-9908 did decrease metastatic burden (i.e., size of metastases) in all organs examined (lungs, bone, heart, liver, kidneys, pancreas and spleen). Based upon this and other studies, the use of CTCE-9908 is promising as an adjuvant therapy for metastatic disease.

Defining the Hallmarks of Metastasis.

Metastasis is the primary cause of cancer morbidity and mortality. The process involves a complex interplay between intrinsic tumor cell properties as well as interactions between cancer cells and multiple microenvironments. The outcome is the development of a nearby or distant discontiguous secondary mass. To successfully disseminate, metastatic cells acquire properties in addition to those necessary to become neoplastic. Heterogeneity in mechanisms involved, routes of dissemination, redundancy of molecular pathways that can be utilized, and the ability to piggyback on the actions of surrounding stromal cells makes defining the hallmarks of metastasis extraordinarily challenging. Nonetheless, this review identifies four distinguishing features that are required: motility and invasion, ability to modulate the secondary site or local microenvironments, plasticity, and ability to colonize secondary tissues. By defining these first principles of metastasis, we provide the means for focusing efforts on the aspects of metastasis that will improve patient outcomes.

Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases.

Acting on a broad spectrum of extracellular, intracellular, and membrane-associated substrates, the matrix metalloproteinases (MMPs) are critical to the biological processes of organisms; when aberrantly expressed, many pathological conditions may be born or exacerbated. The prospect of MMP inhibition for therapeutic benefit in cancer, cardiovascular disease, and stroke is reviewed here. MMP inhibitor (MMPI) development constitutes an important branch of research in both academic and industrial settings and advances our knowledge on the structure-function relationship of MMPs. Targeting MMPs in disease treatment is complicated by the fact that MMPs are indispensable for normal development and physiology and by their multi-functionality, possible functional redundancy or contradiction, and context-dependent expression and activity. This complexity was revealed by previous efforts to inhibit MMP activity in the treatment of cancer patients that yielded unsatisfactory results. This review focuses on MMPI development since the late 90s, in terms of natural products and their derivatives, and synthetic compounds of low molecular mass incorporating specific zinc-binding groups (ZBGs). A few polyphenols and flavonoids that exhibit MMPI activities may have chemopreventive and neuro- and cardiovascular-protective effects. A new generation of potent and selective MMPIs with novel ZBGs and inhibition mechanisms have been designed, synthesized, and tested. Although only one collagenase inhibitor (Periostat, doxycycline hyclate) has been approved by the Food and Drug Administration as a drug for the treatment of periodontal disease, new hope is emerging in the form of natural and synthetic MMPIs for the prevention and treatment of stroke, cardiovascular disease, cancer, and other medical conditions.

Suppression of murine mammary carcinoma metastasis by the murine ortholog of breast cancer metastasis suppressor 1 (Brms1).

The murine ortholog (Brms1) of human breast cancer metastasis suppressor 1 shares 95% identity to the human metastasis suppressor, BRMS1, in amino acid structure. We tested Brms1 for suppression of metastasis of mouse mammary carcinoma cell line 4T1 in syngenic BALB/c mice, using orthotopic (mammary fat pad) injection as well as intravenous injection. As observed for BRMS1, transfection with Brms1 did not inhibit 4T1 primary tumor formation, but significantly suppressed lung colonization. We also show that Brms1 protein interacts with histone deacetylases, indicating involvement of Brms1 in murine Sin3-HDAC complex, like its human counterpart. Thus, because of similarities with its human ortholog, the results suggest that Brms1 will be useful as a model for studying mechanism of action of BRMS1.

Inhibition of enzyme activity of and cell-mediated substrate cleavage by membrane type 1 matrix metalloproteinase by newly developed mercaptosulphide inhibitors.

MT1-MMP (membrane type 1 matrix metalloproteinase, or MMP-14) is a key enzyme in molecular carcinogenesis, tumour-cell growth, invasion and angiogenesis. Novel and potent MMP inhibitors with a mercaptosulphide zinc-binding functionality have been designed and synthesized, and tested against human MT1-MMP and other MMPs. Binding to the MT1-MMP active site was verified by the competitive-inhibition mechanism and stereochemical requirements. MT1-MMP preferred deep P1' substituents, such as homophenylalanine instead of phenylalanine. Novel inhibitors with a non-prime phthalimido substituent had K(i) values in the low-nanomolar range; the most potent of these inhibitors was tested and found to be stable against air-oxidation in calf serum for at least 2 days. To illustrate the molecular interactions of the inhibitor-enzyme complex, theoretical docking of the inhibitors into the active site of MT1-MMP and molecular minimization of the complex were performed. In addition to maintaining the substrate-specificity pocket (S1' site) van der Waals interactions, the P1' position side chain may be critical for the peptide-backbone hydrogen-bonding network. To test the inhibition of cell-mediated substrate cleavage, two human cancer-cell culture models were used. Two of the most potent inhibitors tested reached the target enzyme and effectively inhibited activation of proMMP-2 by endogenous MT1-MMP produced by HT1080 human fibrosarcoma cells, and blocked fibronectin degradation by prostate cancer LNCaP cells stably transfected with MT1-MMP. These results provide a model for mercaptosulphide inhibitor binding to MT1-MMP that may aid in the design of more potent and selective inhibitors for MT1-MMP.

Globular adiponectin enhances invasion in human breast cancer cells.

Every year, a large number of women succumb to metastatic breast cancer due to a lack of curative approaches for this disease. Adiponectin (AdipoQ) is the most abundant of the adipocyte-secreted adipokines. In recent years, there has been an interest in the use of AdipoQ and AdipoQ receptor agonists as therapeutic agents for the treatment of breast cancer. However, while multiple epidemiological studies have previously indicated that low levels of circulating plasma AdipoQ portend poor prognosis in patients with breast cancer, recent studies have reported that elevated expression levels of AdipoQ in breast tissue are correlated with advanced stages of the disease. Thus, the aim of the present study was to clarify the mechanism by which AdipoQ in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. In the present study, the effects of different AdipoQ isoforms on the metastatic potential of human breast cancer cells were investigated. The results revealed that globular adiponectin (gAd) promoted invasive cell morphology and significantly increased the migration and invasion abilities of breast cancer cells, whereas full-length adiponectin (fAd) had no effect on these cells. Additionally, gAd, but not fAd, increased the expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B)-II and intracellular LC3B puncta, which are indicators of autophagosome formation, thus suggesting autophagic induction by gAd. Furthermore, the inhibition of autophagic function by autophagy-related protein 7 knockdown attenuated the gAd-induced increase in invasiveness in breast cancer cells. Therefore, the results of the present study suggested that a specific AdipoQ isoform may enhance breast cancer invasion, possibly via autophagic induction. Understanding the roles of the different AdipoQ isoforms as microenvironmental regulatory molecules may aid the development of effective AdipoQ-based treatments for breast cancer.

Increased autophagic response in a population of metastatic breast cancer cells.

Breast cancer cells are heterogeneous in their ability to invade and fully metastasize, and thus also in their capacity to survive the numerous stresses encountered throughout the multiple steps of the metastatic cascade. Considering the role of autophagy as a survival response to stress, the present study hypothesized that distinct populations of breast cancer cells may possess an altered autophagic capacity that influences their metastatic potential. It was observed that a metastatic breast cancer cell line, MDA-MB-231, that was sensitive to autophagic induction additionally possessed the ability to proliferate following nutrient deprivation. Furthermore, a selected subpopulation of these cells that survived multiple exposures to starvation conditions demonstrated a heightened response to autophagic induction compared to their parent cells. Although this subpopulation maintained a more grape-like pattern in three-dimensional culture compared to the extended spikes of the parent population, autophagic induction in this subpopulation elicited an invasive phenotype with extended spikes. Taken together, these results suggest that autophagic induction may contribute to the ability of distinct breast cancer cell populations to survive and invade.