Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Oct 1.
Published in final edited form as: Cancer Discov. 2014 Feb 10;4(4):466–479. doi: 10.1158/2159-8290.CD-13-0841

Autophagy dependent production of secreted factors facilitates oncogenic RAS-driven invasion

Rebecca Lock 1, Candia M Kenific 1, Andrew M Leidal 1, Eduardo Salas 1, Jayanta Debnath 1
PMCID: PMC3980002  NIHMSID: NIHMS565499  PMID: 24513958

Abstract

The tumor promoting functions of autophagy are primarily attributed to its ability to promote cancer cell survival. However, emerging evidence suggests that autophagy plays other roles during tumorigenesis. Here, we uncover that autophagy promotes oncogenic RAS-driven invasion. In epithelial cells transformed with oncogenic RAS, depletion of autophagy-related genes suppresses invasion in three-dimensional culture, decreases cell motility, and reduces pulmonary metastases in vivo. Treatment with conditioned media from autophagy-competent cells rescues the invasive capacity of autophagy-deficient cells, indicating these cells fail to secrete factors required for RAS-driven invasion. Reduced autophagy diminishes the secretion of the pro-migratory cytokine IL6, which is necessary to restore invasion of autophagy-deficient cells. Moreover, autophagy-deficient cells exhibit reduced levels of MMP2 and WNT5A. These results support a previously unrecognized function for autophagy in promoting cancer cell invasion via the coordinate production of multiple secreted factors.

Keywords: Autophagy, RAS, Invasion, Three-dimensional culture, Secretion, Interleukin 6

INTRODUCTION

The RAS proteins are members of a family of small GTPases critical in mediating cellular responses following activation by upstream extracellular signals, such as growth factors. Oncogenic mutations in RAS, which result in constitutive activation, are found in approximately 30% of human cancers; they are highly prevalent in several carcinomas, including lung, pancreas, and colon (1, 2). Notably, oncogenic RAS drives diverse cellular programs-proliferation, cell survival, migration, invasion and alterations in differentiation-that support tumor initiation and progression. Such mutations present a formidable therapeutic obstacle, because patients harboring mutant KRAS are refractory to most available systemic therapies and exhibit extremely poor survival (2). Hence, identifying new processes to target cancer cells with hyperactive RAS remains a question of immense clinical significance. One such pathway may be macroautophagy (autophagy), a tightly controlled lysosomal degradation process that promotes cell survival during nutrient starvation and stress. Recent evidence indicates that basal autophagy levels are enhanced upon oncogenic RAS activation and support RAS-driven transformation and tumorigenesis (3-7).

The tumor promoting functions of autophagy are largely ascribed to its importance as a survival pathway in response to diverse environmental stresses (8, 9). For example, enhanced autophagy is observed in poorly perfused, hypoxic tumor regions and loss of autophagy is associated with increased necrosis (10). Autophagy also promotes tumor cell survival in response to various cytotoxic and targeted chemotherapies (11). Importantly, studies of oncogenic RAS transformation have revealed that the pro-tumor effects of autophagy are not limited to increased survival of cancer cells under duress; rather, autophagy contributes to the metabolic fitness of the entire tumor population (3-6). Because strong oncogenic insults, such as RAS activation, are marked by profound metabolic alterations that drive both energy production and biosynthetic capacity in rapidly proliferating cells, it has been hypothesized that autophagy maintains key metabolic pathways in RAS-transformed cells. In support, a growing body of work has unveiled a requirement for autophagy in driving proliferation as well as sustaining multiple core metabolic functions in RAS-transformed cells (3-7). These results are not unique to oncogenic RAS activation, as deletion of RB1CC1/FIP200, a mediator of autophagosome initiation, inhibits polyoma middle T driven mammary cancer, due to reduced proliferation and glucose metabolism (12).

In addition to its effects on proliferation and metabolism, oncogenic RAS drives diverse aggressive cellular behaviors that support tumor progression and metastasis; importantly, RAS-transformed epithelial cells exhibit highly invasive behavior associated with an epithelial-to-mesenchymal transition (EMT) (13). Here, in epithelial cells transformed with oncogenic RAS, we demonstrate that autophagy facilitates extracellular matrix (ECM) invasion, tumor cell motility, and pulmonary metastasis in vivo. Using a three-dimensional (3D) culture system, we uncover that autophagy inhibition restricts RAS-driven cell invasion and restores several aspects of normal epithelial architecture, including the polarized deposition of basement membrane and cell-cell junctional integrity. Furthermore, autophagy is required for the production of multiple secreted factors in RAS transformed cells, including interleukin-6 (IL6), matrix metalloproteinase 2 (MMP2), and WNT5A, which altogether facilitate cancer cell invasion.

RESULTS

Autophagy promotes invasion driven by oncogenic RAS in 3D culture

To elucidate how autophagy impacts the cellular behavior of RAS-transformed epithelial cells, we utilized the MCF10A 3D epithelial culture system to interrogate how autophagy affects the growth and morphogenesis of cells expressing oncogenic RAS (14). We generated stable pools of MCF10A human mammary epithelial cells expressing a control vector (BABE) or an oncogenic form of HRAS (HRASV12) that enhances basal autophagy and elicits robust anchorage independent transformation (3). When cultured on laminin-rich ECM, control MCF10A cells formed hollow, spherical acini (Fig. S1A) (15). In contrast, HRASV12 transformed cells produced grossly aberrant structures notable for extensive protrusions that invaded the surrounding extracellular matrix. Individual HRASV12 structures formed these invasive protrusions as early as 3-5 days, ultimately producing disorganized networks of cells intermingled with large cell clusters after 8 days in 3D culture (Fig. 1A and B, left columns). The 3D morphology we observed using HRASV12 MCF10A cells resembles that reported for mouse mammary cells expressing oncogenic RAS and grown in a 3D collagen matrix (16).

Figure 1. Autophagy is required for the formation of invasive protrusions mediated by HRASV12 in 3D culture.

Figure 1

Open in a new tab

(A-B) HRASV12 MCF10A cells stably expressing non-targeting control shRNA (shCNT) or shRNAs against autophagy genes (shATGs) were 3D cultured on Matrigel for the indicated number of days. Representative phase contrast images at the indicated magnifications are shown. Bar, 100μm.

To inhibit autophagy in this experimental system, we stably expressed unique short-hairpin RNAs (shRNA) against two autophagy genes (ATGs)—ATG7 (shATG7-1 and shATG7-2) or ATG12 (shATG12) in MCF10A cells expressing HRASV12. ATG7 or ATG12 knockdown decreased target protein levels, reduced basal and starvation (HBSS) induced autophagy, and increased protein levels of the autophagy substrate p62/SQSTM1 (Fig S1B-E). In 3D culture, the invasive protrusions observed with oncogenic RAS activation were profoundly attenuated in ATG deficient cells. Instead, HRASV12 shATG structures were spherical in morphology, similar to non-transformed BABE controls (Fig. 1A-B). Decreased invasive protrusions following autophagy inhibition were also observed upon stable ATG3 knockdown (shATG3), and upon treatment with chloroquine or bafilomycin A, two lysosomal inhibitors that block the late steps of autophagy (Fig. S1F). Importantly, ATG knockdown in HRASV12 cells did not affect RAS expression or activation associated phosphorylation of the major downstream effector MAPK/ERK (Fig. S1G). Thus, the reduction in 3D invasive protrusions following ATG knockdown is not due to decreased expression or activity of oncogenic RAS.

The disruption of basement membrane integrity is a hallmark of carcinoma invasion in vivo (14). To corroborate whether the protrusions we observed in HRASV12-transformed 3D cultures represented invasive behavior, we first evaluated basement membrane integrity by examining the expression and localization of the basement membrane protein LAMA5 (laminin 5) in HRASV12-derived acini. Consistent with previous reports, control non-transformed MCF10A acini (BABE) displayed polarized deposition of LAMA5 onto the basal surface (Fig. 2A, left panels) (15). In contrast, the expression of HRASV12 resulted in cytosolic accumulation of LAMA5, with no evidence of polarized deposition at the cell-ECM interface. Notably, this aberrant cytosolic staining pattern was especially prominent in the protrusions of HRASV12 cultures. Correlating with the decreased formation of invasive protrusions, ATG knockdown restored polarized LAMA5 secretion; based on this marker, most individual structures in ATG deficient HRASV12 cultures were encompassed by an intact basement membrane (Fig. 2A). Hence, in addition to restricting the formation of invasive protrusions, autophagy inhibition restored polarized basement membrane secretion typically absent in HRASV12 shCNT structures.

Figure 2. Autophagy inhibition in HRASV12 cells restores basement membrane integrity and restricts ECM proteolysis in 3D culture.

Figure 2

Open in a new tab

(A) HRASV12 cells expressing shCNT or shATGs were 3D cultured on Matrigel for 8 days. Structures were fixed and immunostained with antibodies against the basement membrane protein LAMA5 (human specific), counterstained with DAPI to detect nuclei, and imaged by confocal microscopy. Two representative images of each condition are shown. Bar, 50μm. (B) HRASV12 MCF10A cells were 3D cultured on Matrigel containing 25μg/mL fluorescein DQ-collagen IV (DQ-COL4) for 5 days. Structures were fixed, counterstained with phalloidin (to visualize F-actin) and DAPI, and imaged by confocal microscopy. Green fluorescence represents areas of proteolytic cleavage of the DQ-COL4 present in the ECM. Bar, 50μm.

To extend these results, we evaluated ECM proteolytic activity in control and autophagy-deficient HRASV12 cultures by assessing fluorescence emanating from the proteolytic cleavage of dye-quenched collagen IV (COL4). In control non-transformed acini (BABE), we observed a faint ring of fluorescence surrounding each structure, corresponding to COL4 degradation due to the normal outgrowth of acini during 3D morphogenesis. On the other hand, HRASV12 shCNT-expressing structures exhibited high levels of fluorescence that extended well beyond the immediate vicinity of individual structures (Fig. 2B). Notably, streaks of fluorescence connecting adjacent structures were frequently observed in HRASV12 shCNT cultures (Fig. 2B), which resembled the networks of invasive protrusions (Fig 1B). In contrast, HRASV12 shATG-derived structures exhibited a ring-like COL4 degradation pattern that was restricted to the cell-ECM interface, similar to that observed in non-transformed controls (Fig. 2B). Thus, the absence of morphological protrusions in ATG deficient HRASV12 cultures was associated with the restoration of basement membrane integrity and reduced ECM proteolytic activity. Together, these findings corroborate that autophagy supports RAS-driven invasion in 3D culture.

ATG depletion in HRASV12 structures does not promote apoptosis or proliferation arrest in 3D culture

We next evaluated the impact of autophagy inhibition on oncogenic RAS-driven proliferation and cell survival. During normal MCF10A acinar morphogenesis, autophagy inhibition results in the enhanced apoptosis of cells occupying the luminal space (17). To test whether autophagy deficiency similarly impacted apoptosis in HRASV12 structures, we immunostained structures with an antibody against cleaved CASP3 (caspase-3). In contrast to the robust luminal apoptosis observed in control acini (BABE), only isolated cleaved CASP3 positive cells were observed in HRASV12 shCNT structures, consistent with the ability of oncogenic RAS to promote cell survival in 3D culture (Fig 3A). Upon enumerating cleaved CASP3 positive cells from these 3D cultures, we found that ATG knockdown did not significantly impact apoptosis in comparison to shCNT cultures (Fig. 3A). To assess whether autophagy inhibition potentially impacted non-apoptotic death processes, we also stained day 8 3D cultures with ethidium bromide (EtBr), an intravital dye that is incorporated into all dying cells. Whereas acini derived from non-transformed (BABE) cells displayed high levels of EtBr staining corresponding to luminal cell death (Fig. 3B), HRASV12 structures displayed only occasional EtBr cells scattered throughout the structures. Although ATG knockdown in HRASV12 cultures resulted in spherical structures that lacked invasive protrusions, we did not observe any increase in EtBr staining in these cultures (Fig. 3B). Thus, in contrast to normal and oncogenic PIK3CA MCF10A acinar morphogenesis, autophagy inhibition does not promote apoptosis in RAS-transformed 3D structures (17, 18).

Figure 3. Autophagy inhibition in HRASV12 MCF10A structures does not promote apoptosis or proliferation arrest.

Figure 3

Open in a new tab

(A) Left: Two representative images of day 8 3D cultures of BABE and HRASV12 MCF10A cells expressing shCNT or shATGs immunostained with antibody against cleaved CASP3 and counterstained with DAPI to detect nuclei. Bar, 50μm. Right: Quantification of cleaved CASP3 positive cells present within 3D cultures of each indicated cell type (mean +/− s.d., Student’s t-test). (B) Representative phase (top) and corresponding wide-field fluorescence (bottom) images of BABE and HRASV12 cells expressing shCNT or shATGs stained with the intravital dye ethidium bromide (EtBr). Bar, 100μm. (C) Left: Two representative images of day 8 3D cultures of BABE and HRASV12 cells expressing shCNT or shATGs immunostained with antibody against Ki67 and DAPI counterstained. Bar, 50μm. Right: Quantification of Ki67 positive nuclei present within 3D cultures of each indicated cell type (mean +/− s.d., Student’s t-test).

To evaluate the effects of autophagy inhibition on the proliferative capacity of HRASV12 structures, we immunostained cultures with the proliferation marker Ki67 on day 8, a timepoint at which normal MCF10A acini exhibit reduced proliferation (19). As expected, low levels of Ki67 positive cells were observed in BABE structures (Fig 3C, left panels). However, both control and autophagy deficient HRASV12 structures displayed high levels of Ki67 positive cells (Fig 3C). Overall, these results indicate that although autophagy deficiency potently restricts HRASV12 driven invasion, it does not universally suppress the diverse oncogenic effects of HRASV12 in 3D culture, including the ability of activated RAS to inhibit apoptosis and sustain proliferation.

Autophagy supports oncogenic RAS-driven cell migration in vitro and pulmonary metastasis in vivo

Because defects in invasive capacity are often associated with diminished cell motility, we next measured cell migration in autophagy competent and deficient epithelial cells. Upon ATG depletion, HRASV12 MCF10A cells demonstrated an approximately 30% reduction in migratory capacity in a monolayer wound-healing assay of cell migration (Fig. 4A). Similar results were obtained using a transwell migration assay, which demonstrated a significant decrease in migration of ATG knockdown cells (Fig. 4B). We further corroborated these results using MDA-MB-231 cells, a highly migratory, KRAS mutant breast cancer cell line. siRNA-mediated knockdown of either ATG7 or ATG12 in MDA-MB-231 cells resulted in reduced LC3-II formation (Fig. S1H) as well as decreased wound closure (Fig. 4C, left). A similar decrease in MDA-MB-231 migration was also observed in the presence of the lysosomal inhibitor bafilomycin A (Fig. 4C, right). Therefore, in addition to supporting invasion of HRASV12 MCF10A cells in 3D culture, autophagy facilitates the migration of cells expressing oncogenic RAS in monolayer culture. Finally, we utilized an experimental metastasis assay to evaluate whether the effects of autophagy inhibition on invasion and migration correlated with changes in metastatic capacity in vivo; in support, the ability of HRASV12 MCF10A cells to produce pulmonary metastases was reduced upon ATG knockdown (Fig 4D).

Figure 4. ATG knockdown suppresses the motility and reduces the metastatic potential of cells expressing oncogenic RAS.

Figure 4

Open in a new tab

(A) Representative images (left) and quantification (right) of wounding assay on HRASV12 MCF10A cells expressing shCNT or shATGs. Confluent monolayers were scratched and wound width was measured at 0 and 6h after initial wounding to quantify the decrease in scratch width. (mean +/− s.d., Student’s t-test, shCNT n=16, shATG7-2 n=8, shATG12 n=14). Bar, 100μm. (B) Transwell migration of HRASV12 MCF10A cells expressing shCNT or shATGs. 24h after plating, cells that migrated to the bottom of the filter were stained with crystal violet. Results are expressed as the mean crystal violet extracted from stained cells (mean +/− s.d., Student’s t-test, n=9). (C) Wounding assays of MDA-MB-231 cells expressing siATGs or in presence of 10nM bafilomycin A (BafA). Graphs represent the decrease in scratch width at 10h and 9h after initial wounding, respectively (mean +/− s.d., Student’s t-test, siCNT n=16, siATG7 n=16, siATG12 n=10, DMSO n=6, BafA n=6). (D) Representative images (left) and quantification (right) of ZsGreen positive metastatic foci following tail vein injection of ZsGreen expressing HRASV12 shCNT, shATG7-1 or shATG12 cells (mean +/− s.e.m., shCNT n=7, shATG7-1 n=7, shATG12 n=8).

Altered differentiation of HRASV12 MCF10A cells upon autophagy inhibition

Constitutive RAS activation alters epithelial differentiation by driving an epithelial-mesenchymal transition (EMT) (20, 21), a process associated with increased invasive and migratory capacity in vitro and with metastatic capacity in vivo (13). Therefore, we evaluated how autophagy inhibition affects protein expression changes associated with RAS-induced EMT. We isolated BABE, HRASV12 shCNT and HRASV12 shATG expressing cells from day 8 3D cultures and determined the protein expression of a panel of EMT associated genes by immunobloting. In comparison to nontransformed BABE acini, HRASV12 shCNT structures displayed decreased KRT14 (keratin 14), an epithelial marker, and a corresponding increase in the mesenchymal protein VIM (vimentin) (Fig S2A). ATG knockdown reversed these HRASV12-driven changes in differentiation, resulting in an increase in KRT14 protein levels and a corresponding decrease in VIM levels compared to HRASV12 shCNT cells isolated from 3D culture (Fig. S2A). However, autophagy inhibition had minimal effects on other EMT markers that were altered by oncogenic RAS expression. Only a slight increase in CDH1 (E-cadherin) was observed in shATG cells, decreased FN1 (fibronectin) was only observed in shATG7-1 expressing cells, and CDH2 (N-cadherin) levels were unchanged following ATG knockdown (Fig. S2A).

During EMT, cells commonly lose the ability to form cell-cell junctions (22). Therefore, we analyzed the effects of autophagy inhibition on cell-cell junctional integrity in HRASV12 3D structures by immunostaining for CTNNB1 (β-catenin). Normal MCF10A acini (BABE) displayed strong β-catenin staining at cell-cell contacts, indicating intact adherens junctions, whereas the expression of HRASV12 resulted in a near-complete loss of β-catenin junctional staining; in these cultures, only isolated focal areas of junctional β-catenin staining were observed (Fig. S2B). Upon ATG knockdown in HRASV12 structures, both the expression and junctional localization of β-catenin were significantly restored (Fig. S2B). Based on these results, we conclude that autophagy inhibition modulates certain aspects of mesenchymal differentiation in RAS-transformed cells in 3D culture, most notably the suppression of VIM, as well as the restoration of KRT14 expression and epithelial cell-cell contacts. Nonetheless, autophagy deficiency does not broadly suppress RAS-driven EMT.

ATG knockdown in HRASV12 cells inhibits the production of pro-invasive secreted factors in 3D culture

Cell migration and invasion involves the secretion of multiple factors that cooperate to promote motility and to degrade the surrounding ECM (23, 24). To ascertain if defects in RAS-driven invasion observed following autophagy suppression were the result of decreased production of pro-invasive factors, we performed a co-culture assay in which HRASV12 shATG7-1 cells (co-expressing GFP for tracking purposes) were combined with HRASV12 shCNT cells at a ratio of 3:1, respectively. Whereas HRASV12 shATG7-1-GFP cells cultured alone grew as spherical structures (Fig. 5A, left panels), upon co-culture with HRASV12 shCNT cells, HRASV12 shATG7-1-GFP structures became dispersed and formed invasive protrusions (Fig. 5A, right panels). Hence, we hypothesized that factors from neighboring HRASV12 shCNT cells are sufficient to rescue in trans the invasion defect in HRASV12 shATG7-1 cells. To further test this prediction, we grew HRASV12 shATG cells in 3D culture for 3 days and subsequently treated these structures with conditioned media (CM) produced from either BABE or HRASV12 shCNT cultures. HRASV12 shATG structures remained as compact spheres following treatment with BABE CM (Fig. 5B, Fig. S3A). In contrast, CM from HRASV12 shCNT cultures elicited invasive protrusions at 24h following treatment, which became fully evident by 72h (Fig. 5B, Fig. S3A); notably, CM addition did not induce invasion in non-transformed BABE acini (Fig. S3B). Furthermore, basement membrane integrity was lost in HRASV12 shATG cells treated with HRASV12 CM (Fig. 5C). These findings demonstrate that autophagy inhibition in HRASV12 cells inhibits the production of secreted factors required for RAS-driven invasion in 3D culture.

Figure 5. ATG knockdown in HRASV12 cells inhibits the production of pro-invasive secreted factors in 3D culture.

Figure 5

Open in a new tab

(A) 3D co-culture of HRASV12 shATG7-1 with HRASV12 shCNT cells rescues invasion of HRASV12 shATG7-1 cells. HRASV12 shATG7-1 cells expressing GFP were cultured for 8 days in 3D either alone (left) or together with HRASV12 shCNT cells expressing an empty vector (BABE). Structures were imaged by phase contrast and wide-field fluorescence microscopy or fixed, counterstained with phalloidin (to visualize F-actin) and DAPI and imaged by confocal microscopy. Phase: Bar, 100μm. Confocal: Bar, 50 μm. (B) HRASV12 MCF10A cells expressing shATGs were cultured in 3D for 3d and subsequently treated with BABE or HRASV12 shCNT conditioned media (CM). Representative phase contrast images at 24h and 72h following the addition of CM. Bar, 100μm. (C) 3D cultures of HRASV12 MCF10A cells expressing shATGs were treated with BABE or HRASV12 shCNT CM for 72h; thereafter, cultures were fixed and immunostained with an antibody against LAMA5 (human specific) to detect basement membrane and DAPI counterstained. Two representative images per condition are shown. Bar, 50μm.

Diminished secretion of IL6 contributes to reduced invasion in autophagy deficient HRASV12 cells

During RAS-induced senescence, ATG depletion inhibits IL6 production following acute oncogenic RAS activation in IMR90 fibroblasts, indicating autophagy supports the production of IL6 in response to oncogenic RAS activation (25). Because IL6 has been demonstrated to support RAS-driven tumorigenesis, promote migration and invasion, and also drive epithelial-mesenchymal transition (26-28), we tested whether IL6 levels were altered in HRASV12 shATG 3D cultures. Analysis of IL6 in conditioned media collected from 3D cultures by ELISA indicated a significant reduction in secreted IL6 levels in HRASV12 shATG-expressing cultures compared to HRASV12 shCNT cultures (Fig. 6A). Furthermore, this decrease in secreted IL6 was not the result of reduced IL6 gene expression; in fact, qPCR analysis revealed that IL6 transcript levels in HRASV12 shATG cells were increased, rather than decreased, in comparison to HRASV12 shCNT cells (Fig 6B). Notably, studies of RAS-induced senescence similarly demonstrated that autophagy deficient cells exhibit reduced IL6 protein levels due to impaired translation, rather than transcription (25, 29). In contrast, we uncovered that ATG depletion did not attenuate IL6 protein levels in RAS-transformed cells grown in 3D culture (Fig 6C). These results suggest that autophagy facilitates IL6 secretion during HRASV12 3D morphogenesis.

Figure 6. Autophagy supports IL6 secretion necessary for oncogenic RAS-driven invasion in 3D culture.

Figure 6

Open in a new tab

(A) Levels of IL6 in conditioned media collected on day 6 from 3D cultures of the indicated cell types. (mean +/− s.d., ANOVA, BABE n=3, HRASV12 n=5). (B) IL6 expression levels normalized to GAPDH in cells collected from day 8 3D cultures. (mean relative to BABE +/− s.d., Student’s t-test, n=3). (C) IL6 protein levels in day 8 3D cultures from the indicated cell types. (D) Representative phase contrast images of HRASV12 shATG 3D cultures treated for 48h with BABE CM (top) or with HRASV12 shCNT CM containing an IL6 function-blocking antibody (bottom) or IgG control antibody (middle). Bar, 100μm. (E) Representative phase contrast images of HRASV12 shATG 3D cultures grown in the presence or absence of 200ng/mL recombinant human IL6 for 7d. Bar, 100μm.

To ascertain the functional significance of these results, we interrogated whether IL6 was necessary for HRASV12-driven invasion in 3D culture. First, we treated HRASV12 shATG structures with HRASV12 shCNT CM in the presence versus absence of an IL6 function-blocking antibody. The addition of IL6 function-blocking antibody attenuated the ability of HRASV12 shCNT CM to promote invasive protrusions in HRASV12 shATG cultures, whereas an IgG isotype control had no effect (Fig. 6D). In parallel, we tested how exogenous recombinant human IL6 (rhIL6) treatment affected HRASV12 shATG cells during 3D morphogenesis. rhIL6 addition did not affect nontransformed BABE acini (Fig. S3C) but partly restored invasion in HRASV12 shATG cultures, resulting in large globular structures, increased invasive protrusions, and loss of basement membrane integrity (Fig. 6E, S3D-E). Also, rhIL6 addition partially reversed the effects of autophagy inhibition on KRT14 and VIM expression in HRASV12 shATG7 cells (Fig. S3F). Hence, our results suggest that autophagy promotes efficient IL6 secretion by HRASV12 cells in 3D culture, which is necessary for invasion.

Autophagy facilitates MMP2 and WNT5A expression by HRASV12 cells in 3D culture

In addition to identifying a defect in IL6 production following ATG knockdown, we performed a qPCR array to measure the expression levels of genes involved in EMT and invasion, and identified WNT5A and MMP2 as two candidate factors whose expression was upregulated in HRASV12 cells relative to BABE cells but potently suppressed upon autophagy inhibition. qPCR analysis of cells collected from 3D cultures confirmed a 2-fold decrease in MMP2 and WNT5A expression in HRASV12 shATG cells compared to HRASV12 shCNT (Fig. 7A and B). Notably, we also evaluated the effects of rhIL6 treatment on MMP2 and WNT5A expression in shATG7-1 cultures and found this was not sufficient to rescue expression, indicating that regulation of these factors was independent of IL6 (Fig. S3G).

Figure 7. WNT5A and MMP2 are reduced following autophagy inhibition in 3D culture.

Figure 7

Open in a new tab

(A-B) RNA was isolated from BABE, HRASV12 shCNT, and HRASV12 shATG cells cultured in 3D for 8 days. Expression levels of MMP2 and WNT5A were determined by qPCR and normalized to an internal control GAPDH. Results represent the mean relative to BABE +/− s.d. (MMP2, n=4; WNT5A, n=3; Student’s t-test). (C) Conditioned media was collected from BABE, HRASV12 shCNT and HRASV12 shATG cells grown in 3D culture. Activity levels of MMP9 and MMP2 in the conditioned media were determined by zymography. (D) HRASV12 shCNT cells were grown in the absence (top) or presence (bottom) of 25μm Arp-100. Left: Structures were imaged on day 8 by phase contrast microscopy. Right: Representative confocal images of structures immunostained with anti-phospho-ERM (P-ERM) to detect cell borders and counterstained with DAPI. Bars, 100μm. (E) BABE, HRASV12 shCNT, and HRASV12 shATG cells were collected from 3D culture on day 8, lysed, and protein levels of WNT5A were determined by immunoblot analysis. (F) HRASV12 shATG7-1 cells were grown in 3D for 8 days in the absence (top) or presence (bottom) of 500ng/mL WNT5A. Left: Representative phase contrast images. Right: Representative confocal images of structures immunostained with anti-phospho-ERM to detect cell borders and counterstained with DAPI. Bars, 100μm.

Because these secreted factors have been implicated in cell migration and invasion, we further evaluated whether their decreased expression following ATG knockdown also contributed to the reduced invasive potential of HRASV12 shATG cells. First, we utilized gelatin zymography to assess MMP2 activity in CM from 3D cultures. MMP2 activity was enhanced in HRASV12 cells compared to non-transformed (BABE) controls, and upon ATG knockdown in HRASV12 cells, this activity was reduced (Fig. 7C). The increase in MMP2 expression and secretion following constitutive RAS activation was necessary for RAS-driven invasion, as addition of an MMP2 inhibitor, Arp-100, was sufficient to inhibit the formation of invasive protrusions in HRASV12 3D cultures (Fig. 7D). Furthermore, the decrease in WNT5A expression correlated with a decrease in WNT5A protein levels in HRASV12 shATG cells isolated from 3D culture (Fig. 7E). Moreover, the addition of recombinant WNT5A to HRASV12 shATG7-1 3D cultures promoted the dissociation of cells within the structures and enhanced the formation of invasive protrusions (Fig. 7F). Thus, in addition to IL6, autophagy facilitates the production of multiple secreted pro-migratory and invasive factors that support RAS-driven invasion in 3D culture.

DISCUSSION

Our results delineate a previously unrecognized function for autophagy in facilitating oncogenic RAS-driven invasion and migration. Using a 3D culture system, we demonstrate that suppression of autophagy in HRASV12 MCF10A cells restricts the formation of invasive protrusions, restores basement membrane integrity, and attenuates ECM proteolysis. In addition, autophagy inhibition diminishes cell migration in vitro and pulmonary metastasis in vivo. Upon treatment with conditioned media produced from autophagy-competent HRASV12 cells, invasion is completely restored in autophagy-deficient HRASV12 cultures, indicating that autophagy mediates the production of secreted factors that drive invasion in oncogenic cells. In further support, we uncover that autophagy inhibition elicits the coordinate reduction of multiple molecules favoring invasion. Overall, these findings expand our understanding of how autophagy supports cancer progression.

Although autophagy inhibition suppresses invasion in 3D culture, it does not ubiquitously revert oncogenic RAS-driven changes in cell behavior. Indeed, MAPK activation remains unaltered following autophagy inhibition in this 3D culture model and moreover, the oncogenic activation of RAS continues to disrupt fundamental aspects of 3D morphogenesis in autophagy deficient cells. First, autophagy inhibition does not alter the ability of HRASV12 to suppress apoptosis in 3D culture. Moreover, autophagy inhibition does not suppress proliferation in HRASV12 3D cultures; rather, the spherical structures from HRASV12 ATG knockdown cells remain highly proliferative over extended periods. Remarkably, both others and we have shown that ATG depletion reduces soft agar growth and attenuates the proliferation of RAS-transformed cells grown in monolayer (3-5, 30); hence, the absence of proliferative suppression in this 3D culture model may be context dependent. These results also differ from those obtained in KRAS mutant mouse cancer models in which genetic ATG deletion impairs proliferation and, in certain cases, enhances apoptosis (4, 6, 7). Certain reasons may explain these differences. First, we have only reduced ATGs using RNAi, rather than genetically eliminated these proteins. Second, the experiments here are significantly shorter duration in comparison to autophagy deficient K-Ras tumor growth in vivo.

Although previous studies have demonstrated that autophagy supports the invasion of glioblastoma cells, the mechanistic underpinnings remain unclear (31, 32). Cell invasion requires the production and secretion of factors that stimulate migration and degrade the surrounding ECM (24). Upon treatment of autophagy depleted HRASV12 cells with conditioned media produced from their autophagy competent counterparts, the ability to form invasive protrusions is completely restored, suggesting that autophagy is required for the efficient production of secreted factors that promote invasion and migration of HRASV12 cells. Notably, conditioned media treatment does not promote invasion in nontransformed BABE cells, indicating oncogenic Ras pathway activation is still required for invasion.

Importantly, we identify IL6 as one critical factor whose secretion is ATG dependent; our results substantiate that this pro-invasive cytokine is necessary to restore invasion in autophagy-deficient HRASV12 cells. They also point to a specific role for autophagy in facilitating IL6 secretion; upon ATG knockdown, RAS-transformed cells fail to secrete IL6 into the conditioned media, yet both IL6 transcription and translation remain intact. These results differ from recent studies of oncogenic RAS-mediated senescence, in which reduced IL6 secretion in autophagy-deficient cells is proposed to be secondary to decreased protein synthesis (25, 29).

Though traditionally viewed as an autodigestive process, growing evidence suggests new roles for autophagy in both conventional and unconventional secretion (33). Indeed, a genetic role for ATGs has been implicated in: 1) unconventional secretion of proteins lacking N-terminal ER signal sequences (34-37); 2) efficient egress of secretory lysosomes (38, 39); and 3) conventional secretion of growth factors (40, 41). Further dissecting how autophagy directs the secretion of IL6 and other factors during RAS transformation remains an important topic for future study. Remarkably, IL6 re-addition only partially restores invasion and mesenchymal differentiation in HRASV12 autophagy-deficient cultures, indicating other factors promote invasion. In support, these cells exhibit reduced levels of other pro-invasive molecules, including WNT5A and MMP2. In contrast to reduced IL6 secretion, which is likely a proximal event following ATG knockdown, these changes in WNT5A and MMP2 result from decreased gene expression, indicating that autophagy inhibition produces broader transcriptional changes contributing to reduced invasion by HRASV12 cells.

Recently, the deletion of RB1CC1/FIP200, a gene mediating autophagosome initiation, was demonstrated to reduce lung metastases in the MMTV-PyMT breast cancer model. However, since RB1CC1 deletion profoundly restricted primary tumor growth, it was unclear whether decreased metastasis was secondary to reduced primary tumor burden (12). In addition, although liver-specific deletion of ATG7 or ATG5 initiates the development of benign adenomas, these tumors are unable to progress to adenocarcinomas, suggesting that autophagy is required for advanced tumor progression (42, 43). Here, in epithelial cells transformed with oncogenic RAS, we demonstrate that defective autophagy results in decreased invasion and migration, which correlates with the reduced ability to metastasize in vivo. Although our results do not rule out potentially important functions for autophagy in disseminated cell survival or outgrowth at foreign tissue sites, they delineate new roles for autophagy in the control of secretion during carcinoma progression.

EXPERIMENTAL PROCEDURES

Cell lines

MCF10A cells were obtained from the American Tissue Culture Collection (ATCC) and cultured as previously described (44). MDA-MB-231 cells were obtained from the ATCC and cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. Cell lines were passaged for less than 6 months following resuscitation and were not authenticated.

Three dimensional culture assays

MCF10A overlay 3D culture was performed as previously described (44). As indicated, the following reagents were added to cultures: 500ng/mL WNT5A (R&D Systems), 200ng/mL IL6 (Peprotech), 25 μg/mL anti-IL6 function blocking antibody (R&D Systems), 25 μg/mL IgG control antibody (BD Biosciences), 25μM Arp-100 (Santa Cruz Biotechnology), 5μM chloroquine diphosphate salt (Sigma), and 5nM bafilomycin A (Sigma). For the 3D ECM degradation assay, human DQ-COL4 (Invitrogen) was mixed with Matrigel to a final concentration of 25μg/mL prior to plating. To collect cells for immunoblotting and RNA isolation, cultures were incubated with 0.25% Trypsin/EDTA at 37°C for 10 min to dissociate cells from surrounding matrix and create a single cell suspension. Cells were resuspended in media containing 20% serum and washed twice with PBS to remove residual Matrigel.

For co-culture assays, shATG7-1 was expressed in HRASV12 cells stably expressing pBABEhygro GFP. This GFP-labeled “target” cell line was then cultured in isolation or combined with unlabelled (pBABEhygro) HRASV12 shCNT cells at a ratio of 3:1 with total cell number kept constant at 7,500 cells/well. For conditioned media (CM) experiments, HRASV12 shATG expressing cells were grown in 3D culture for 3d; subsequently, the media was replaced with conditioned media harvested from BABE or HRASV12 shCNT MCF10A cells grown in 3D culture for 6-8 days. When indicated, 25 μg/mL anti-IL6 function blocking or IgG isotype control antibody was added to the CM.

Wounding Assay

Cells were grown to confluence in 3.5 cm dishes and incubated overnight in assay media lacking EGF for MCF10A cells or DMEM+2% FBS for MDA-MB-231 cells. Wound healing was performed in the presence of 2μg/ml mitomycin C (Sigma). Cells were wounded with a 200μl pipette tip and imaged at time of wounding (0 h) and the indicated time points. Average wound widths were measured at each time point and decreases in wound width were calculated by subtracting the average width at the final time point from the average width at 0 h using MetaMorph Software (v6.0).

Transwell Assay

Cells were starved overnight in assay media lacking EGF and then plated at 1.0×105 in the top chamber of an 8μm Transwell filter in assay media lacking EGF. The bottom chamber was filled with assay medium containing 5 ng/ml EGF. Cells were allowed to migrate for 24 h, after which the top of each filter was cleared of cells. Cells attached to the bottom of the filter were fixed and stained with crystal violet. Crystal violet was extracted with 10% acetic acid and the absorbance was measured at 600 nm.

Experimental metastasis assay

For experimental metastasis assays, cells were infected with pHIV-ZsGreen (Addgene, Cambridge, MA, plasmid 18121). 1.0×106 HRASV12 shCNT, shATG7-1, and shATG12 cells stably expressing ZsGreen were injected into the tail vein of NOD/SCID mice. After 140 days, whole lungs were fixed and imaged to detect the number of ZsGreen positive foci per lung. All animal experiments were conducted in accordance with approved UCSF IACUC protocols.

IL6 ELISA

Day 5 3D cultures were washed twice with PBS and cultured for 18 h in serum-free media. Conditioned media was collected, and total protein levels were determined by BCA assay (Thermo Scientific) to normalize samples. IL6 levels were measured using the Quantikine High Sensitivity ELISA kit (R&D Systems).

Statistical analyses

Each experiment was repeated at least three independent times. GraphPad Prism software (v5.0b) was used for generation of graphs and statistical analysis. P values were determined by Student’s t-test or ANOVA as stated.

Supplementary Material

1
2
3
4

SIGNIFICANCE.

Our results delineate a previously unrecognized function for autophagy in facilitating oncogenic RAS-driven invasion. We demonstrate that an intact autophagy pathway is required for the elaboration of multiple secreted factors favoring invasion, including IL6.

ACKNOWLEDGEMENTS

Grant support to JD includes the NIH (R01CA126792 and −S1), the DOD BCRP (W81XWH-11-1-0130 and W81XWH-12-1-0505), Samuel Waxman Cancer Research Foundation, and the California TRDRP (18XT-0106). RL was supported by a DOD BCRP Predoctoral Award (W81XWH-08-1-0759) and CMK by a Genentech Fellowship, University of California CRCC Fellowship, and NIH F31CA167905.

Abbreviations

(ECM)

Extracellular matrix

(ATG)

autophagy-related gene

(EMT)

epithelial-mesenchymal transition

Footnotes

Conflicts of interest: The authors have no conflicts of interest to disclose.

CONTRIBUTIONS

RL, CMK, AML and JD designed the experiments. RL, CMK, AML, ES and JD carried out the experiments as well as analyzed and interpreted results. JD supervised the overall study. RL and JD wrote the paper with comments from the other authors.

REFERENCES

  • 1.Schubbert S, Shannon K, Bollag G. Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer. 2007;7:295–308. doi: 10.1038/nrc2109. [DOI] [PubMed] [Google Scholar]
  • 2.Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer. 2011;11:761–74. doi: 10.1038/nrc3106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Lock R, Roy S, Kenific CM, Su JS, Salas E, Ronen SM, et al. Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation. Mol Biol Cell. 2011;22:165–78. doi: 10.1091/mbc.E10-06-0500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, et al. Pancreatic cancers require autophagy for tumor growth. Genes Dev. 2011;25:717–29. doi: 10.1101/gad.2016111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G, et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 2011;25:460–70. doi: 10.1101/gad.2016311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Guo JY, Karsli-Uzunbas G, Mathew R, Aisner SC, Kamphorst JJ, Strohecker AM, et al. Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev. 2013;27:1447–61. doi: 10.1101/gad.219642.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Rosenfeldt MT, O’Prey J, Morton JP, Nixon C, MacKay G, Mrowinska A, et al. p53 status determines the role of autophagy in pancreatic tumour development. Nature. 2013;504:296–300. doi: 10.1038/nature12865. [DOI] [PubMed] [Google Scholar]
  • 8.Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev. 2011;25:1999–2010. doi: 10.1101/gad.17558811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Chen N, Debnath J. Autophagy and tumorigenesis. FEBS Lett. 2010;584:1427–35. doi: 10.1016/j.febslet.2009.12.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer cell. 2006;10:51–64. doi: 10.1016/j.ccr.2006.06.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss WA, Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res. 2011;17:654–66. doi: 10.1158/1078-0432.CCR-10-2634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wei H, Wei S, Gan B, Peng X, Zou W, Guan JL. Suppression of autophagy by FIP200 deletion inhibits mammary tumorigenesis. Genes Dev. 2011;25:1510–27. doi: 10.1101/gad.2051011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer. 2009;9:265–73. doi: 10.1038/nrc2620. [DOI] [PubMed] [Google Scholar]
  • 14.Debnath J, Brugge JS. Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer. 2005;5:675–88. doi: 10.1038/nrc1695. [DOI] [PubMed] [Google Scholar]
  • 15.Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS. The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell. 2002;111:29–40. doi: 10.1016/s0092-8674(02)01001-2. [DOI] [PubMed] [Google Scholar]
  • 16.Oft M, Peli J, Rudaz C, Schwarz H, Beug H, Reichmann E. TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes & development. 1996;10:2462–77. doi: 10.1101/gad.10.19.2462. [DOI] [PubMed] [Google Scholar]
  • 17.Fung C, Lock R, Gao S, Salas E, Debnath J. Induction of Autophagy during Extracellular Matrix Detachment Promotes Cell Survival. Mol Biol Cell. 2008;19:797–806. doi: 10.1091/mbc.E07-10-1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Chen N, Eritja N, Lock R, Debnath J. Autophagy restricts proliferation driven by oncogenic phosphatidylinositol 3-kinase in three-dimensional culture. Oncogene. 2012 doi: 10.1038/onc.2012.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Muthuswamy SK, Li D, Lelievre S, Bissell MJ, Brugge JS. ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini. Nature cell biology. 2001;3:785–92. doi: 10.1038/ncb0901-785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Thiery JP. Epithelial-mesenchymal transitions in development and pathologies. Current opinion in cell biology. 2003;15:740–6. doi: 10.1016/j.ceb.2003.10.006. [DOI] [PubMed] [Google Scholar]
  • 21.Shin S, Dimitri CA, Yoon SO, Dowdle W, Blenis J. ERK2 but not ERK1 induces epithelial-to-mesenchymal transformation via DEF motif-dependent signaling events. Molecular cell. 2010;38:114–27. doi: 10.1016/j.molcel.2010.02.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Xu J, Lamouille S, Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 2009;19:156–72. doi: 10.1038/cr.2009.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Scheel C, Eaton EN, Li SH, Chaffer CL, Reinhardt F, Kah KJ, et al. Paracrine and autocrine signals induce and maintain mesenchymal and stem cell States in the breast. Cell. 2011;145:926–40. doi: 10.1016/j.cell.2011.04.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer. 2003;3:362–74. doi: 10.1038/nrc1075. [DOI] [PubMed] [Google Scholar]
  • 25.Young AR, Narita M, Ferreira M, Kirschner K, Sadaie M, Darot JF, et al. Autophagy mediates the mitotic senescence transition. Genes Dev. 2009;23:798–803. doi: 10.1101/gad.519709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Ancrile B, Lim KH, Counter CM. Oncogenic Ras-induced secretion of IL6 is required for tumorigenesis. Genes & development. 2007;21:1714–9. doi: 10.1101/gad.1549407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Leslie K, Gao SP, Berishaj M, Podsypanina K, Ho H, Ivashkiv L, et al. Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation. Breast Cancer Res. 2010;12:R80. doi: 10.1186/bcr2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Sullivan NJ, Sasser AK, Axel AE, Vesuna F, Raman V, Ramirez N, et al. Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human breast cancer cells. Oncogene. 2009;28:2940–7. doi: 10.1038/onc.2009.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Narita M, Young AR, Arakawa S, Samarajiwa SA, Nakashima T, Yoshida S, et al. Spatial coupling of mTOR and autophagy augments secretory phenotypes. Science (New York, NY.) 2011;332:966–70. doi: 10.1126/science.1205407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kim MJ, Woo SJ, Yoon CH, Lee JS, An S, Choi YH, et al. Involvement of autophagy in oncogenic K-Ras-induced malignant cell transformation. J Biol Chem. 2011;286:12924–32. doi: 10.1074/jbc.M110.138958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Galavotti S, Bartesaghi S, Faccenda D, Shaked-Rabi M, Sanzone S, McEvoy A, et al. The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells. Oncogene. 2013;32:699–712. doi: 10.1038/onc.2012.111. [DOI] [PubMed] [Google Scholar]
  • 32.Macintosh RL, Timpson P, Thorburn J, Anderson KI, Thorburn A, Ryan KM. Inhibition of autophagy impairs tumor cell invasion in an organotypic model. Cell Cycle. 2012;11:2022–9. doi: 10.4161/cc.20424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Deretic V, Jiang S, Dupont N. Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation. Trends Cell Biol. 2012;22:397–406. doi: 10.1016/j.tcb.2012.04.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Duran JM, Anjard C, Stefan C, Loomis WF, Malhotra V. Unconventional secretion of Acb1 is mediated by autophagosomes. The Journal of cell biology. 2010;188:527–36. doi: 10.1083/jcb.200911154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Manjithaya R, Anjard C, Loomis WF, Subramani S. Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation. The Journal of cell biology. 2010;188:537–46. doi: 10.1083/jcb.200911149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Manjithaya R, Subramani S. Autophagy: a broad role in unconventional protein secretion? Trends Cell Biol. 2011;21:67–73. doi: 10.1016/j.tcb.2010.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1beta. EMBO J. 2011;30:4701–11. doi: 10.1038/emboj.2011.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Ushio H, Ueno T, Kojima Y, Komatsu M, Tanaka S, Yamamoto A, et al. Crucial role for autophagy in degranulation of mast cells. J Allergy Clin Immunol. 2011;127:1267–76 e6. doi: 10.1016/j.jaci.2010.12.1078. [DOI] [PubMed] [Google Scholar]
  • 39.DeSelm CJ, Miller BC, Zou W, Beatty WL, van Meel E, Takahata Y, et al. Autophagy proteins regulate the secretory component of osteoclastic bone resorption. Dev Cell. 2011;21:966–74. doi: 10.1016/j.devcel.2011.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, Komiya K, et al. Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab. 2008;8:325–32. doi: 10.1016/j.cmet.2008.08.009. [DOI] [PubMed] [Google Scholar]
  • 41.Jung HS, Chung KW, Won Kim J, Kim J, Komatsu M, Tanaka K, et al. Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia. Cell Metab. 2008;8:318–24. doi: 10.1016/j.cmet.2008.08.013. [DOI] [PubMed] [Google Scholar]
  • 42.Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S, et al. Autophagy-deficient mice develop multiple liver tumors. Genes Dev. 2011;25:795–800. doi: 10.1101/gad.2016211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Inami Y, Waguri S, Sakamoto A, Kouno T, Nakada K, Hino O, et al. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J Cell Biol. 2011;193:275–84. doi: 10.1083/jcb.201102031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods (San Diego, Calif.) 2003;30:256–68. doi: 10.1016/s1046-2023(03)00032-x. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS565499-supplement-1.doc (68.5KB, doc)
2
NIHMS565499-supplement-2.tif (911.7KB, tif)
3
NIHMS565499-supplement-3.tif (1.8MB, tif)
4
NIHMS565499-supplement-4.tif (1.3MB, tif)

RESOURCES