Lysosomal Transmembrane Protein LAPTM4B Promotes Autophagy and Tolerance to Metabolic Stress in Cancer Cells

Author Manuscript Published OnlineFirst on October 28, 2011; DOI: 10.1158/0008-5472.CAN-11-0940 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Lysosomal transmembrane protein LAPTM4B promotes autophagy and tolerance to metabolic stress in cancer cells

Yang Li1, Qing Zhang1, Ruiyang Tian1, Qi Wang1, Jean J. Zhao1, J. Dirk Iglehart1, 2, Zhigang

Charles Wang1, Andrea L. Richardson1, 2

1 Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 USA

2 Brigham and Women’s Hospital, Harvard Medical School, Boston MA 02115 USA

Correspondence: [email protected] or [email protected]

Running Title: LAPTM4B promotes autophagy and cell tolerance to stress

Precis: Overexpression of a lysosomal protein implicated in chemoresistance and breast cancer is shown to promote autophagy and inhibit lysosome-mediated death pathways.

Key Words: breast cancer, autophagy, lysosome-mediated death, metabolic stress

This work supported by Susan G. Komen For the Cure (AR, ZW, YL, RT), Breast Cancer Research Foundation (JDI, AR, ZW), Terri Brodeur Breast Cancer Foundation (YL), and Friends of DFCI (YL).

The authors have no conflicts of interest to disclose.

Word count: 4997; Figures: 6

Abstract

Amplification of chromosome 8q22, which includes the gene for lysosomal-associated transmembrane protein LAPTM4B, has been linked to de novo anthracycline resistance in primary breast cancers with poor prognosis. LAPTM4B overexpression can induce cytosolic retention of anthracyclines and to decrease drug induced DNA damage. In this study, we tested the hypothesis that LAPTM4B may contribute to tumor cell growth or survival in the absence of a chemotherapeutic exposure. In mammary cells, LAPTM4B protein was localized in lysosomes where its depletion increased membrane permeability, pH, cathepsin release and cellular apoptosis. Loss of LAPTM4B also inhibited later stages of autophagy by blocking maturation of the autophagosome, thereby rendering cells more sensitive to nutrient deprivation or hypoxia. Conversely, enforced overexpression of

LAPTM4B promoted autophagic flux and cell survival during in vitro starvation and stimulated more rapid tumor growth in vivo. Together, our results indicate that

LAPTM4B is required for lysosome homeostasis, acidification, and function, and that

LAPTM4B renders tumor cells resistant to lysosome-mediated cell death triggered by environmental and genotoxic stresses.

Introduction

Chemoresistance is often an acquired characteristic of recurrent tumors either induced or selected by exposure to a drug. Alternatively, de novo drug resistance can occur due to intrinsic

features of the primary tumor which are selected by contributing a proliferative or survival advantage to tumor cells. The possible mechanisms of chemoresistance may include altered drug uptake, intracellular distribution, efflux and turnover. Lysosomal retention of drugs is associated with drug resistance and lysosomal concentration of anthracyclines is thought to increase drug efflux and decrease drug nuclear localization, thereby preventing effective chemotherapy- induced DNA damage (1).

Lysosomes are organelles that contain hydrolytic enzymes such as proteases, nucleases and lipases. Lysosomal-membrane permeabilization (LMP) can pose a threat to cellular homeostasis through release of lysosomal contents and is a recognized trigger of cell death (2).

For example, a selective and limited release of lysosomal cathepsins B or D may activate the mitochondrial cell death pathway by either generating reactive oxygen species and lipid mediators or triggering mitochondrial outer membrane permeabilization. Cathepsin B is a major mediator of apoptotic pathways activating the pro-apoptotic protein Bid by truncation. Truncated

Bid (tBid) translocates to mitochondria to active the caspase cascade and promotes apoptosis.

Cathepsin D is an aspartate protease that directly activates caspase (3, 4). The current knowledge of the proteins on lysosomal membranes critical for affecting or regulating these various lysosomal functions is limited.

Autophagy is a conserved lysosome-mediated intracellular trafficking pathway that degrades and recycles intracellular components (5). Autophagy is also a homeostatic mechanism that regulates metabolism and energy production and may be up regulated in response to a variety of cell stresses (5-7). As cancers develop and disseminate, the process of autophagy

(autophagy flux) may be up-regulated to support tumor cell survival and allow tumors to adapt to these stresses (8-10). In addition, autophagy has been shown to promote cell survival in

response to chemotherapeutic agents (11-13). Conversely, too much autophagy may catabolize essential components resulting in autophagic cell death (14, 15). Autophagy is regulated by a signaling cascade involving mammalian target of rapamycin (mTOR) pathway inhibition, the autophagy proteins (Atgs), and two ubiquitin-like conjugation systems (16-20). During autophagy initiation, a portion of the cytosol is surrounded by a cisternal membrane, the phagophore (21), that closes to form a double-membraned vesicle, the autophagosome (22).

During autophagosome formation, cytosolic LC3 is cleaved by a protease and then conjugated to phosphatidylethanolamine to form autophagosomal membrane-associated LC3II (23); the level of

LC3II correlates with the number of autophagosomes (24, 25). After their formation, autophagosomes fuse with endosomes to form amphisomes (26, 27) and then with lysosomes to form autolysosomes where sequestered material is degraded (5, 28-30). Blockade of autophagosome maturation and fusion with the lysosomal compartment results in accumulation of autophagosomes and interruption in the flux of material through the autophagic pathway (31).

Autophagic flux can be monitored by measuring the level of substrates normally degraded by autophagy such as p62/SQSTM1 (25). The p62 protein is a ubiquitin-binding scaffold protein that binds directly to LC3 and is itself degraded in autolysosomes (32). Inhibition of autophagic degradation results in an increase in p62 levels and increased autophagy flux is indicated by decreased p62 levels (33). Many molecules have been implicated in these later stages of autophagosome maturation (30).

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B) is a putative novel oncogene (34, 35) frequently amplified and overexpressed in primary treatment-naive breast cancers (36). Like other LAPTM family members, LAPTM4B protein has a lysosome localization motif (34) and co-localizes with markers of late endosomes and lysosomes (37).

Increased expression of LAPTM4B has been demonstrated in hepatocellular carcinoma (HCC), and lung, ovarian, uterine, and gastric cancers (35, 38-40). Overexpression of the LAPTM4B-35 isoform in hepatocellular carcinoma cell lines promotes apoptosis resistance in vitro and growth and metastasis of HCC xenografts in mice (41). We reported overexpression of LAPTM4B in primary breast tumors is associated with resistance to chemotherapy, specifically anthracyclines, and may serve as a predictive biomarker for distant recurrence in patients treated with adjuvant chemotherapy (36). By sequestering drug in cytoplasmic compartment and enhancing efflux of drugs from cancer cells, overexpression of LAPTM4B decreases nuclear localization of drug and drug-induced DNA damage and thereby reduces drug effectiveness (36, 42). However, the potential mechanisms by which LAPTM4B promotes tumor growth and survival in treatment- naive cancers is not well studied.

This study tests the effect of modulating LAPTM4B expression on cell survival under various stress conditions including nutrient deprivation, pH alteration, hypoxia, chemotherapy exposure, and in vivo tumor growth. Our results demonstrate that high expression of LAPTM4B inhibits lysosome-mediated death pathways, promotes autophagy, and leads to stress tolerance, thereby enhancing tumor cell growth and survival. LAPTM4B may be an important new therapeutic target for inhibiting cancer growth or sensitizing tumors to chemotherapy.

Material and Methods

Cell lines, siRNA transfection and gene transfer. Breast cell lines BT549 and MDA-MB-468 were obtained from American Type Culture Collection and were authenticated by Promega

PowerPlex 1.2 short tandem repeat profiling. Tert-immortalized human mammary epithelial cells

(HMEC) were from Clontech and provided by the Zhao lab. BT549 cancer cells have

amplification and overexpression of LAPTM4B (43) and were used for siRNA knockdown experiments. MDA-MB-468 (MDA468) cancer cells have normal DNA copy number and expression of LAPTM4B (43) and were chosen for LAPTM4B-transfection for overexpression and tumor xenograft experiments. Cell culture condition, siRNA transfection, expression construct carrying LAPTM4B cDNA, and stable transfer of this construct into MDA468 were performed as described (36).

Analysis of lysosomal membrane permeabilization (LMP). Cells were transfected with control and LAPTM4B siRNA. After 48 hours, control and LAPTM4B expression-modified cells were incubated with FITC-labeled dextrans of different molecular weights from 4, 40, and

70KDa (Sigma) for 2 or 12 hours, followed by wash out of excess dextran with culture medium, examination on a Zeiss HAL100 fluorescence microscope and photographed with a Zeiss camera.

Tumorigenicity assay. Two groups of mice were injected with LAPTM4B expression- manipulated MDA468 cells or the control counterpart cells into the inguinal mammary fat pads.

Mice were routinely monitored for health and tumor size; mice were sacrificed when tumors reached 2 centimeters. Tumor growth rates were measured and compared between LAPTM4B- manipulated and control tumors. LAPTM4B expression level and the autophagosome marker

LC3 were measured by immunoblot in tumor lysates prepared from xenograft tumors.

Statistical analysis. Difference in cell viability, tumor size, and ratios of LC3II/LC3I, or of p62/actin between control and testing groups was evaluated by student t-test and p-values derived from this test were used to determine the significance of the difference.

Additional methods are described in Supplementary Material.

Results

LAPTM4B localizes to lysosomes and down regulation of LAPTM4B triggers lysosome membrane permeabilization

LAPTM4B is a member of a family of proteins which contain a lysosome targeting motif at the

C terminus (34). To determine lysosome localization in mammary epithelial cells, we examined

HMEC cells stably expressing exogenous six-His epitope tagged LAPTM4B by immunofluorescence microscopy and demonstrated expression of His-LAPTM4B in the lysosomal compartment defined by the lysosome-marker Lysotracker DND-99 (Fig. S1A).

To understand how LAPTM4B might modulate lysosome function, we investigated the relationship between LAPTM4B expression and LMP. LMP was evaluated by measuring endocytic uptake and release of a non-digestible fluid-phase substrate, fluorescein isothiocyanate

(FITC)-dextran. In BT549 breast cancer cells transfected with a scramble control siRNA, after 2 hours of exposure to FITC-dextran, the fluorescent dextran particles were detected in a punctate cytoplasmic pattern consistent with lysosomes and were retained in the lysosomal compartment

12 hours later (Fig 1A; Fig. S2A-B). In BT549 cells transfected with LAPTM4B-specific siRNA, the fluorescence pattern was similar to control cells at 2 hours; however, after 12 hours the 4 kDa and 40 kDa FITC-dextran molecules were in a diffuse distribution throughout the cell consistent with release from the lysosomal compartment (Fig. 1B; Fig.S2C-D). The distribution of the larger molecular weight 70 kDa FITC-dextrans were mostly unchanged after 12 hours (Fig.

1B, lower panels). These results suggest that LAPTM4B exerts lysosome-stabilizing properties to retain low and intermediate molecular weight macromolecules. Doxorubicin is small molecule with a molecular weight of approximately 580 Dalton. As shown in Fig.1C, after 2 hours of drug exposure doxorubicin autofluorescence is co-localized with Lysotracker in both the control- treated and LAPTM4B-siRNA treated BT549 cells consistent with initial uptake of drug into

lysosomes. Doxorubicin is retained in the lysosomal compartment 24 hours later in the control siRNA-treated or untreated BT549 cells (Fig. 1D, upper panels; Fig.S1B). However, in the

BT549 cells treated with LAPTM4B-siRNA, after 12-24 hours doxorubicin has been redistributed and predominantly co-localizes with nuclear DAPI staining (Fig. 1D, lower panels).

This result suggests that knockdown of LAPTM4B does not completely abolish the uptake of doxorubicin by cytoplasmic organelles, but significantly weakens the capability of lysosomes to retain the drug, resulting in its more rapid release and redistribution to the nucleus.

Doxorubicin is also a weak base that can be trapped in acidified lysosomes. To examine whether LAPTM4B expression has an effect on lysosomal pH, we utilized a pH sensing dye,

LysoSensor Green DND-189, which is taken up into lysosomes and fluoresces with greater intensity in lower pH. As shown in Fig. 2A, BT549 cells treated with LAPTM4B-siRNA have a higher lysosomal pH (lower fluorescent intensity) compared to control cells. The degree of increase in lysosomal pH is similar to BT549 cells treated with 50 μM Chloroquine (CQ), a lysosomotropic agent that raises the lysosomal pH (Fig. 2B). However, SKBR3 breast cancer cells with forced stable overexpression of LAPTM4B had similar lysosomal pH compared to control cells (Fig. 2C). To determine if change in lysosomal pH alone could account for increased permeability of the lysosome, we treated BT549 cells with 50 M CQ to raise lysosomal pH and found no alteration in the uptake of dextran at 2 hours and dextran was not released into the cytosol at 12 hours (Fig. S2E-F). These results suggest that LAPTM4B may not directly regulate lysosomal pH but the increase of lysosomal pH in LAPTM4B knockdown cells may be a secondary effect of increased LMP resulting in deacidification of the lysosome.

Knockdown of LAPTM4B by siRNA provokes lysosomal-mediated programmed cell death through induction of cathepsin release

The maintenance of lysosomal membrane integrity is important for cell survival. LMP leads to cathepsin release followed by caspase activation. This process initiates apoptosis which triggers further lysosomal destabilization to induce lysosomal-mediated cell death in a positive feed back loop (2). To determine if LAPTM4B knockdown led to cathepsin release from lysosomes, we performed immunoblot analysis for cathepsins on cell cytosol fractions of BT549 tumor cells after transfection with control or LAPTM4B-specific siRNA. Depletion of LAPTM4B resulted in appearance of cathepsin B and cathepsin D in the cytosol of cells (Fig. 3A) and resulted in Bid truncation, caspase 3 activation and PARP cleavage (Fig. 3B) consistent with initiation of apoptosis. Pan inhibitors of cathepsins (EST and pepstatin A) protected BT549 cells from

LAPTM4B knockdown-induced Bid truncation and apoptosis. The pan-caspase inhibitor z-

VAD-FMK was sufficient to abrogate caspase 3 activation but did not prevent Bid truncation.

Treatment with 50 μM CQ alone was not sufficient to induce cathepsin release and resulted in no added effect to LAPTM4B knockdown on cathepsin release or apoptosis induction (Fig. S3, lanes 4 and 8). However, the release of cathepsin B and D and the activation of caspase 3 were more pronounced in cells also treated with the DNA damaging drug, doxorubicin (Fig. 3A-B;

Fig. S3, lanes 2 and 6). There was no potentiation of cathepsin release or caspase 3 activation in cells also treated with the microtubule stabilizing agent taxol (Fig. S3, lanes 3 and 7), consistent with previous studies showing that taxol-induced apoptosis may be independent of caspase 3 and 9 activation(44). These results suggest that a certain level of LAPTM4B is required to prevent lysosome-mediated initiation of apoptosis. This requirement is more notable in the setting of additional cellular insult, such as exposure to certain chemotherapy agents.

LAPTM4B over-expression promotes autophagy

Autophagy plays a critical survival role by supporting energy requirements and sustaining viability under adverse conditions and may also promote resistance to chemotherapy induced genotoxic stress. To determine if modulation of LAPTM4B expression has an effect on autophagy, we examined markers of autophagosome maturation and flux during starvation- induced autophagy in BT549 breast cancer cells with or without depletion of LAPTM4B. In parental BT549 cells (with inherent overexpression of LAPTM4B), autophagy was induced by starvation as indicated by the appearance of punctate LC3 staining in the cytoplasm. Many of the LC3-puncta co-localized with the lysosome marker LAMP2 indicating normal autophagosome maturation and fusion to lysosomes (Fig. 4A). In contrast, in cells in which

LAPTM4B has been depleted by siRNA, starvation resulted in marked cytoplasmic accumulation of enlarged LC3-positive autophagosomes, some of which were not co-localized with LAMP2-positive lysosomes (Fig. 4B). This suggests that depletion of LAPTM4B results in a block in autophagosome-lysosome fusion and blocked autolysosome formation.

We next analyzed autophagy in these cells by immunoblotting for LC3II and p62. siRNA depletion of LAPTM4B had no effect on the level of LC3II in BT549 cells grown in nutrient rich medium, but resulted in significant increase in the level of LC3II when cells were stressed by serum starvation (Fig. 4C and Fig. S4A). LC3II is an indicator of autophagosome number which can increase due to increased autophagosome formation or from a block in autophagosome maturation or both (31). To determine if autophagy flux was increased or blocked, we measured levels of the autophagy substrate, p62. In control cells, serum starvation increased autophagy flux as indicated by lower p62 levels in the serum starved cells compared to

cells grown in nutrient rich medium. However, in cells depleted of LAPTM4B, there was no change in the level of p62 after serum starvation, indicating a block in starvation-induced autophagy flux (Fig. 4C and Fig. S4B). The effect of knockdown of LAPTM4B on LC3II and p62 levels was similar to what was observed in serum starved cells treated with CQ. In conjunction with the immunofluorescence findings, these results suggest that LAPTM4B is required for the later stages of autophagy maturation in which autophagosomes are fused with lysosomes to form autolysosomes.

Abortive accumulation of autophagasomes may act as a cell death messenger to trigger caspase dependent or independent cell death. We found an increase in cell death as indicated by caspase 3 activation and cleaved PARP in LAPTM4B-depleted cells with starvation-induced aggregation of autophagasomes (Fig. 4C). Although down regulation of LAPTM4B alone may trigger LMP and to a lesser extent caspase activation, the combination of LMP and blocked autophagy results in more pronounced caspase activation and cell death.

In a complimentary experiment, stable over expression of LAPTM4B in MDA468 cells with inherently low levels of LAPTM4B, resulted in greater starvation-induced autophagy as indicated by higher levels of LC3II and lower levels of p62 (Fig. 4D and Fig. S4C and D). This effect was blocked by the autolysosome inhibitor chloroquine. These results are consistent with the notion that over-expression of LAPTM4B promotes increased autophagy flux in cancer cells exposed to metabolic stress.

Over expression of LAPTM4B promotes tumor cell survival and resistance to apoptosis induced by low serum concentration or glucose deprivation

The previous experiments suggest that overexpression of LAPTM4B may result in decreased sensitivity of tumor cells to insults that trigger LMP, lysosome-mediated programmed cell death, or induce autophagy. We examined the effect of modulating LAPTM4B expression levels on cell survival in cells exposed to various environmental stressors. In BT549 cells with amplification and over-expression of LAPTM4B, siRNA depletion of LAPTM4B resulted in approximately

50% decrease (P = 0.0267) in viable cells when cultured in nutrient-rich medium. Depletion of

LAPTM4B resulted in a similar or more pronounced decrease in viable cells when grown in low serum or low glucose medium (P = 0.001 and 0.0260, respectively; Fig. 5A) MDA468 breast cancer cells have inherently low normal levels of LAPTM4B expression. MDA468 cells with forced over-expression of LAPTM4B had similar cell viability to parental cells when cultured in nutrient-rich media (P = 0.105). These results suggest that a certain level of LAPTM4B may be required for tumor cell survival, but its over-expression does not directly promote in vitro cell growth under normal culture conditions. However, the cells with over-expression of LAPTM4B showed higher cell viability than control cells when cultured in low serum or low glucose medium (P = 0.0256 and 0.0960, respectively; Fig. 5B). As shown in Fig. 5C, the decreased cell viability of BT549 cells after knockdown of LAPTM4B was associated with increased activation of caspase 3 and PARP cleavage. Similarly, the enhanced survival of MDA468 cells overexpressing LAPTM4B in nutrient deprived conditions was associated with much lower levels of active caspase 3 and PARP cleavage. These results support our hypothesis that high expression of LAPTM4B potentiates the growth and survival of breast cancer cells under metabolic stress.

Next, we tested whether modulation of LAPTM4B affects survival of cells exposed to other types of stress. As shown in Fig. S5A and B, expression levels of LAPTM4B had no effect on cell viability when cells were cultured in acidified medium. Exposure to hypoxic stress

showed variable results. Forced overexpression of LAPTM4B in MDA468 cells had no significant effect on cell viability of cells grown in low oxygen (Fig. S5C). However, BT549 with LAPTM4B-depletion were more sensitive to hypoxia and had reduced viability compared to parental BT549 cells that over-express LAPTM4B (Fig. S5D).

We further examined the effects of LAPTM4B modulation on tumor growth and survival in an in vivo xenograft model. MDA468 cells with stable expression of either a control vector or a vector driving LAPTM4B over-expression were inoculated into the cleared mammary fat pad of nude mice. After two months, we found that the tumor xenografts that over-expressed

LAPTM4B grew much faster than control xenografts (t-test P = 0.033, Fig. 6A). Examination of the xenograft tumor explants demonstrated that each continued to over-express LAPTM4B compared to control xenograft tumors (Fig. 6B); the presence of the smaller proteolytic fragments indicates proper delivery of LAPTM4B to the lysosomes (37). We examined tumor lysates for markers of autophagy by immunoblot (Fig. 6C). LAPTM4B-overexpressing xenografts demonstrated higher LC3II / LC3I ratio (P = 0.0040, Fig. 6D), decreased p62 levels (P

= 0.0363, Fig. 6E), and higher LC3II / LC3I / p62 ratio (P = 0.0074, Fig. 6F) consistent with increased autophagic flux. This result suggests that LAPTM4B over-expression promotes tumor growth in vivo, perhaps by promoting greater tolerance to stress through increased induction of autophagy.

Discussion

The cancer gene LAPTM4B is one of two genes amplified and overexpressed from chromosome

8q22 which predicts for de novo anthracycline resistance and metastatic recurrence in breast

cancer patients (36). Our previous work suggested one mechanism by which LAPTM4B may promote chemotherapy resistance is by retention of anthracyclines in a cytoplasmic compartment thereby preventing nuclear drug localization and drug-induced DNA damage. The frequent occurrence of 8q amplification and overexpression of LAPTM4B in treatment-naive cancers raised the possibility that LAPTM4B may provide a growth or survival advantage to tumor cells in the absence of a therapy challenge, resulting in its selection and upregulation in primary

(untreated) cancers. In this study, we provide evidence that LAPTM4B is localized in lysosomes of mammary cells and promotes lysosome membrane stability. Decreased expression of

LAPTM4B leads to increased LMP, increase in lysosomal pH, lysosomal release of cathepsins and lysosome-mediated cell death. This effect is more pronounced when cells are exposed to anthracycline chemotherapy but not when exposed to a taxane, consistent with the increased sensitivity to anthracyclines in tumors with low LAPTM4B expression (36). We find

LAPTM4B plays a critical role in autophagy and insufficient levels of LAPTM4B results in a failure of autophagosome-lysosome fusion causing a block in autolysosome formation and decreased autophagy flux. In contrast, overexpression of LAPTM4B in breast cancer cells that have normal DNA copy number of 8q22 and express normal levels of LAPTM4B results in increased autophagy flux. These observations suggest that the proper function of LAPTM4B is required for lysosome-mediated autophagy maturation. A recent study showed that transient over-expression of LAPTM4B without coordinate increase of the expression of its partner

MCOLN1 in retinal epithelial cells led to increased LC3II levels due to an accumulation of autophagosomes(37). This finding could result from increased autophagy induction or a block in autophagy flux, but is consistent with the notion that appropriate levels of LAPTM4B are crucial to proper lysosome-mediated autophagy.

Fusion of lysosome with autophagosome is a critical step for autophagy maturation. Rab7,

SKD1, Lamp2, UVRAG and Rebicon have been shown to be essential for this process (5, 29, 30,

37, 45, 46). Frequent frameshift mutations have been identified in the UVRAG gene in colon and gastric cancers (47); however, no significant aberrations in autophagy were found in cancer cells carrying this mutation (48). To the best of our knowledge, LAPTM4B represents the first gene crucial for autophagy maturation and flux that is amplified in cancer (36, 49) and is associated with treatment resistance and poor clinical outcome in various cancers(34, 36, 39-41,

49). Metadherin was recently shown to induce autophagy in different cell lineages by increasing expression of atg5 and activating AMP kinase (50). Interestingly, the gene for metadherin,

MTDH, is neighbor to LAPTM4B and is co-amplified with LAPTM4B in cancers. We propose that the coordinate amplification of the two genes may activate simultaneously the two major stages of the autophagy pathway and thereby enhance tumor survival.

Tumors frequently experience elevated metabolic stress from nutrient and oxygen deprivation due to insufficient angiogenesis (8). The high metabolic demands of cell proliferation and altered metabolism provide additional tumor cell-intrinsic stress. Thus, tumors may be more dependent on survival mechanisms such as autophagy to maintain growth or to disseminate (10, 51). We show the roles of LAPTM4B in limiting lysosome-mediated cell death and promoting autophagy have significant survival effects in cancer cells, including greater resistant to nutrient deprivation, hypoxia, and chemotherapy-induced genotoxic stress. We further demonstrate LAPTM4B pro-survival effect is associated with greater in vivo tumor growth. The new knowledge from this study contributes to a better understanding of the molecular mechanisms of lysosome-mediated drug resistance and the particular dependency of tumors on lysosomal function for cell survival. As normal tissues are seldom under nutrient

deficiency, there may be a significant therapeutic window for inhibiting LAPTM4B in tumors that over-express this gene and are dependent on it for survival. Over-reliance of cancer cells on

LAPTM4B for tolerance to stress may represent the ‘Achilles heel’ for malignancies that have amplified this gene and provide a new therapeutic strategy for targeting these cancers.

ACKNOWLEDGMENTS

We thank Lisa Cameron (DF/HCC confocal core facility) and members of the Richardson-Wang lab for their advice and assistance. We are very grateful to Dr. William Kaelin for allowing us to use the hypoxia chamber in his laboratory. This work was supported by the Susan G. Komen for the Cure (KG080939), the Terri Brodeur Breast Cancer Foundation, and The Friends of Dana-

Farber Cancer Institute. Support also came from the NCI Harvard SPORE in Breast Cancer (2

P50 CA89393-06), a DOD Concept Award (BC053041), and the Breast Cancer Research

Foundation.

References

1. Larsen, A. K., Escargueil, A. E., and Skladanowski, A. Resistance mechanisms associated with altered intracellular distribution of anticancer agents. Pharmacol Ther 2000; 85: 217-229. 2. Johansson, A. C., Appelqvist, H., Nilsson, C., Kagedal, K., Roberg, K., and Ollinger, K. Regulation of apoptosis-associated lysosomal membrane permeabilization. Apoptosis 2010; 15: 527-540. 3. Baumgartner, H. K., Gerasimenko, J. V., Thorne, C., Ashurst, L. H., Barrow, S. L., Chvanov, M. A., et al. Caspase-8-mediated apoptosis induced by oxidative stress is independent of the intrinsic pathway and dependent on cathepsins. Am J Physiol Gastrointest Liver Physiol 2007; 293: G296-307. 4. Conus, S., Perozzo, R., Reinheckel, T., Peters, C., Scapozza, L., Yousefi, S., et al. Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation. J Exp Med 2008; 205: 685-698. 5. Burman, C. and Ktistakis, N. T. Autophagosome formation in mammalian cells. Semin Immunopathol 2010; 32: 397-413.

6. He, C. and Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 2009; 43: 67-93. 7. Rabinowitz, J. D. and White, E. Autophagy and metabolism. Science 2010; 330: 1344-1348. 8. Jin, S. and White, E. Role of autophagy in cancer: management of metabolic stress. Autophagy 2007; 3: 28-31. 9. 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. 10. Kenific, C. M., Thorburn, A., and Debnath, J. Autophagy and metastasis: another double-edged sword. Curr Opin Cell Biol 2010; 22: 241-245. 11. Fan, Q. W., Cheng, C., Hackett, C., Feldman, M., Houseman, B. T., Nicolaides, T., et al. Akt and autophagy cooperate to promote survival of drug-resistant glioma. Sci Signal 2010; 3: ra81. 12. Rubin, B. P. and Debnath, J. Therapeutic implications of autophagy-mediated cell survival in gastrointestinal stromal tumor after treatment with imatinib mesylate. Autophagy 2010; 6: 1190-1191. 13. Levine, B. Cell biology: autophagy and cancer. Nature 2007; 446: 745-747. 14. Scarlatti, F., Granata, R., Meijer, A. J., and Codogno, P. Does autophagy have a license to kill mammalian cells? Cell Death Differ 2009; 16: 12-20. 15. Eisenberg-Lerner, A., Bialik, S., Simon, H. U., and Kimchi, A. Life and death partners: apoptosis, autophagy and the cross-talk between them. Cell Death Differ 2009; 16: 966-975. 16. Klionsky, D. J., Cregg, J. M., Dunn, W. A., Jr., Emr, S. D., Sakai, Y., Sandoval, I. V., et al. A unified nomenclature for yeast autophagy-related genes. Dev Cell 2003; 5: 539-545. 17. Yorimitsu, T. and Klionsky, D. J. Autophagy: molecular machinery for self-eating. Cell Death Differ 2005; 12 Suppl 2: 1542-1552. 18. Wullschleger, S., Loewith, R., and Hall, M. N. TOR signaling in growth and metabolism. Cell 2006; 124: 471-484. 19. Hanada, T., Noda, N. N., Satomi, Y., Ichimura, Y., Fujioka, Y., Takao, T., et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem 2007; 282: 37298-37302. 20. Sou, Y. S., Waguri, S., Iwata, J., Ueno, T., Fujimura, T., Hara, T., et al. The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol Biol Cell 2008; 19: 4762-4775. 21. Seglen, P. O., Gordon, P. B., and Holen, I. Non-selective autophagy. Semin Cell Biol 1990; 1: 441-448. 22. Juhasz, G. and Neufeld, T. P. Autophagy: a forty-year search for a missing membrane source. PLoS Biol 2006; 4: e36. 23. Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J 2000; 19: 5720-5728. 24. Klionsky, D. J., Cuervo, A. M., and Seglen, P. O. Methods for monitoring autophagy from yeast to human. Autophagy 2007; 3: 181-206. 25. Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew, D. S., et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4: 151-175. 26. Razi, M., Chan, E. Y., and Tooze, S. A. Early endosomes and endosomal coatomer are required for autophagy. J Cell Biol 2009; 185: 305-321.

27. Stromhaug, P. E. and Seglen, P. O. Evidence for acidity of prelysosomal autophagic/endocytic vacuoles (amphisomes). Biochem J 1993; 291 (Pt 1): 115- 121. 28. Dunn, W. A., Jr. Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J Cell Biol 1990; 110: 1935-1945. 29. Eskelinen, E. L. Maturation of autophagic vacuoles in Mammalian cells. Autophagy 2005; 1: 1-10. 30. Mehrpour, M., Esclatine, A., Beau, I., and Codogno, P. Overview of macroautophagy regulation in mammalian cells. Cell Res 2010; 20: 748-762. 31. Rubinsztein, D. C., Cuervo, A. M., Ravikumar, B., Sarkar, S., Korolchuk, V., Kaushik, S., et al. In search of an "autophagomometer". Autophagy 2009; 5: 585- 589. 32. Pankiv, S., Clausen, T. H., Lamark, T., Brech, A., Bruun, J. A., Outzen, H., et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007; 282: 24131-24145. 33. Bjorkoy, G., Lamark, T., Pankiv, S., Overvatn, A., Brech, A., and Johansen, T. Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol 2009; 452: 181-197. 34. Shao, G. Z., Zhou, R. L., Zhang, Q. Y., Zhang, Y., Liu, J. J., Rui, J. A., et al. Molecular cloning and characterization of LAPTM4B, a novel gene upregulated in hepatocellular carcinoma. Oncogene 2003; 22: 5060-5069. 35. Kasper, G., Vogel, A., Klaman, I., Grone, J., Petersen, I., Weber, B., et al. The human LAPTM4b transcript is upregulated in various types of solid tumours and seems to play a dual functional role during tumour progression. Cancer Lett 2005; 224: 93-103. 36. Li, Y., Zou, L., Li, Q., Haibe-Kains, B., Tian, R., Li, Y., et al. Amplification of LAPTM4B and YWHAZ contributes to chemotherapy resistance and recurrence of breast cancer. Nat Med 2010; 16: 214-218. 37. Vergarajauregui, S., Martina, J. A., and Puertollano, R. LAPTMs regulate lysosomal function and interact with mucolipin 1: new clues for understanding mucolipidosis type IV. J Cell Sci 2010; 124: 459-468. 38. Liu, Y., Zhang, Q. Y., Qian, N., and Zhou, R. L. Relationship between LAPTM4B gene polymorphism and susceptibility of gastric cancer. Ann Oncol 2007; 18: 311- 316. 39. Yang, H., Xiong, F. X., Lin, M., Yang, Y., Nie, X., and Zhou, R. L. LAPTM4B-35 overexpression is a risk factor for tumor recurrence and poor prognosis in hepatocellular carcinoma. J Cancer Res Clin Oncol 2010; 136: 275-281. 40. Meng, F. L., Yin, M. Z., Song, H. T., Yang, H., Lou, G., and Zhou, R. L. LAPTM4B-35 overexpression is an independent prognostic marker in endometrial carcinoma. Int J Gynecol Cancer 2010; 20: 745-750. 41. Yang, H., Xiong, F., Wei, X., Yang, Y., McNutt, M. A., and Zhou, R. Overexpression of LAPTM4B-35 promotes growth and metastasis of hepatocellular carcinoma in vitro and in vivo. Cancer Lett 2010; 294: 236-244. 42. Li, L., Wei, X. H., Pan, Y. P., Li, H. C., Yang, H., He, Q. H., et al. LAPTM4B: a novel cancer-associated gene motivates multidrug resistance through efflux and activating PI3K/AKT signaling. Oncogene 2010; 29: 5785-5795. 43. Neve, R. M., Chin, K., Fridlyand, J., Yeh, J., Baehner, F. L., Fevr, T., et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 2006; 10: 515-527.

44. Ofir, R., Seidman, R., Rabinski, T., Krup, M., Yavelsky, V., Weinstein, Y., et al. Taxol-induced apoptosis in human SKOV3 ovarian and MCF7 breast carcinoma cells is caspase-3 and caspase-9 independent. Cell Death Differ 2002; 9: 636-642. 45. Liang, C., Lee, J. S., Inn, K. S., Gack, M. U., Li, Q., Roberts, E. A., et al. Beclin1- binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nat Cell Biol 2008; 10: 776-787. 46. Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N., et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages. Nat Cell Biol 2009; 11: 385-396. 47. Kim, M. S., Jeong, E. G., Ahn, C. H., Kim, S. S., Lee, S. H., and Yoo, N. J. Frameshift mutation of UVRAG, an autophagy-related gene, in gastric carcinomas with microsatellite instability. Hum Pathol 2008; 39: 1059-1063. 48. Knaevelsrud, H., Ahlquist, T., Merok, M. A., Nesbakken, A., Stenmark, H., Lothe, R. A., et al. UVRAG mutations associated with microsatellite unstable colon cancer do not affect autophagy. Autophagy 2010; 6: 863-870. 49. Hu, G., Chong, R. A., Yang, Q., Wei, Y., Blanco, M. A., Li, F., et al. MTDH activation by 8q22 genomic gain promotes chemoresistance and metastasis of poor- prognosis breast cancer. Cancer Cell 2009; 15: 9-20. 50. Bhutia, S. K., Kegelman, T. P., Das, S. K., Azab, B., Su, Z. Z., Lee, S. G., et al. Astrocyte elevated gene-1 induces protective autophagy. Proc Natl Acad Sci U S A 2010; 107: 22243-22248. 51. White, E. and DiPaola, R. S. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 2009; 15: 5308-5316.

Figure legends

Figure 1. Suppression of LAPTM4B expression triggers lysosome membrane permeabilization. Merged fluorescence analysis for dextran (FITC, green) and nuclear staining

(DAPI, blue) to show intracellular distribution of 4 kDa (top row), 40 kDa (middle row), or 70 kDa (lower row) dextran particles in A. BT549 cells transfected with a scrambled control siRNA and B. BT549 cells transfected with LAPTM4B-specific siRNA. The times after dextran exposure are indicated on top. C. and D. Merged fluorescence analysis for doxorubicin (drug autofluorescence, red), lysosomes (Lysotracker DND-26, green) and nuclear staining (DAPI, blue) in BT549 cells transfected with control siRNA (Ctrl, upper row) and with LAPTM4B- specific siRNA (siRNA, lower row) after 2 or 24 hours of exposure to doxorubicin.

Figure 2. LAPTM4B depletion changes acidification of lysosomes. Cell counts (y-axis) by fluorescence intensity (x-axis, log scale) of cells stained with LysoSensor Green DND-189. Cell treatments are indicated as follows: purple filled area, cells stained with PBS (1% BSA); green line, cells transfected with siRNA-control or empty vector; pink line, BT549 cells transfected with siRNA-LAPTM4B (panel A) or treated with 50M chloroquine for 24 hours (panel B), or

SKBR3 cells with forced stable expression of LAPTM4B (panel C).

Figure 3. Suppression of LAPTM4B induces cathepsin-dependent cleavage of Bid,

Caspase-3 and PARP in breast cancer cells that over express LAPTM4B. A. BT549 cells transfected with a scrambled control siRNA (Ctrl) or LAPTM4B-specific siRNA (L4B siRNA) incubated in medium alone (---) or with 1μM doxorubicin (Doxo). Proteins (100 g per lane) from cytosol preparations were fractionated on SDS-PAGE; blots were probed with anti-

Cathepsin B, D, and anti-actin. B. BT549 cells pretreated with cathepsin inhibitors, pepstatin A and EST (Cath inh), or pan-caspase inhibitor z-VAD-fmk (Casp inh) then transfected with control or LAPTM4B-specific siRNA and treated with medium alone (---) or 1 μM doxorubicin

(Doxo). Proteins (100 g per lane) from cell lysates were fractionated on SDS-PAGE; blots were probed with anti-Bid, anti-caspase 3, anti-PARP, and anti-actin antibodies.

Figure 4. Effects of modulating LAPT4B expression on autophagy maturation and flux A. and B. Merged immunofluorescence analysis for autophagosomes (anti-LC3, red), lysosomes

(anti-LAMP2, green), and nuclear staining (DAPI, blue) in BT549 cells transfected with A. control scramble siRNA and B. LAPTM4B-specific siRNA. Cells were cultured in nutrient

rich medium (Normal, upper panels of A. and B.) or in low-serum medium (Starvation, lower panels of A. and B.) C. Immunoblot of control siRNA-treated BT549 cells in medium alone (---) or with chloroquine (CQ) or LAPTM4B-siRNA treated BT549 in medium alone (si-L4B). D.

Immunoblot of MDA468 cells transfected with control vector cultured with medium alone (---) or chloroquine (CQ) or MDA468 cells transfected LAPTM4B-expression vector cultured in medium alone (L4B) or with chloroquine (L4B+CQ). C. and D. Cells were cultured in nutrient rich medium (Normal, left side) or low serum medium (Starvation, right side). Proteins (100 g per lane) from cell lysates were fractionated on SDS-PAGE; blots were probed with anti-LC3, anti-p62, anti-active caspase 3, anti-PARP, and anti-actin as indicated.

Figure 5. Effects of modulating LAPTM4B expression on tolerance to nutrient stress.

Nutrient and glucose-dependent cell survival curves in A. BT549 cells transfected with scramble control ( ) and LATPM4B-specific siRNA ( ) and B. MDA468 cells transfected with control GFP vector ( ), and LAPTM4B vector ( ). The ratio of viable cells at each time point vs. time zero are indicated on y-axis (mean of triplicates ± s.d.). P-values from t-test for difference between control and LAPTM4B-manipulated cells are indicated as follows: * P < 0.1,

** P < 0.03, *** P = 0.001. C. Immunoblot of MDA468 cells transfected with control GFP vector (Ctrl), and LAPTM4B vector (L4B) and BT549 cells transfected with scramble control

(Ctrl) and LATPM4B-specific siRNA (si-L4B). Cell lines are indicated along the top. Proteins

(100 g per lane) from cell lysates were fractionated on SDS-PAGE; blots were probed with anti-caspase 3, anti-PARP, and anti-actin antibodies. Cells were cultured in nutrient rich, low serum, and low glucose medium as indicated above the panels or lanes.

Figure 6. Over expression of LAPTM4B promotes increased autophagy and faster tumor growth in vivo A. Tumor growth of MDA468 expressing control vector ( ) or LAPTM4B vector ( ) in immunodeficient mice. B. Immunoblotting for His epitope tagged LAPTM4B in tumor explant lysates derived from MDA468 cells expressing a control vector (left lane) or

LAPTM4B vector (right lanes). Full length LAPTM4B (black arrow) and smaller proteolytic fragments (white arrows) indicate delivery of LAPTM4B to lysosomes. C. Immunoblotting for

LC3 and p62 in tumor explant lysates derived from cells expressing a control vector (left lanes, n

= 4) or LAPTM4B vector (right lanes, n = 5). D. E. F. Quantification of LC3II to LC3I ratio, p62 to actin ratio and LC3II/LC3I to p62 ratio, respectively. Data represent mean + standard deviation of MDA468 xenograft tumors expressing a control vector (white bars, n = 4) or

LAPTM4B vector (black bars, n = 5).

A B Ctrl siRNA L4B siRNA Ctrl siRNA L4B siRNA --- Doxo --- Doxo

Cathepsin B Cath inh + Cath inh +

Doxo --- Doxo --- Cath InhCasp inh Casp inh + DoxoCath inhCasp inh Casp inh + Doxo

Cathepsin D Bid tBid

Actin Active Caspase 3

cl PARP

Actin

A Control LC3 LAMP2 DAPI Merge

Normal

Starvation

B siRNA-LAPTM4B LC3 LAMP2 DAPI Merge

Normal

Starvation

C Normal Starvation D Normal Starvation L4B+ L4B+ --- CQ si-L4B --- CQ si-L4B --- L4B CQ CQ --- L4B CQ CQ

LC3 LC3I I LC3 LC3II II

P62 P62

active actin Caspase 3

cl PARP

Actin

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2011; DOI: 10.1158/0008-5472.CAN-11-0940 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 5 A Nutrient rich Low serum Low glucose ** *** ** 6 6 6 ctrl siRNA-L4B 4 4 4 BT549 2 2 2 Ratio of viable cells Ratio of viable cells Ratio of viable cells 0 0 0 01234 01234 01234 Days Days Days

B Nutrient rich Low serum Low glucose 8 8 ** 8 * ctrl 6 6 6 LAPTM4B

4 4 4 MDA468 2 2 2

Ratio of viable cells 0 Ratio of viable cells 0 Ratio of viable cells 0 0 123 4 0123 4 0 123 4 Days Days Days C MDA468 BT549

Nutrient richLow serumLow glucoseNutrient richLow serumLow glucose Ctrl L4B Ctrl L4B Ctrl L4B Ctrl si-L4B Ctrl si-L4B Ctrl si-L4B Active Caspase 3

cl PARP

Actin

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 28, 2011; DOI: 10.1158/0008-5472.CAN-11-0940 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Figure 6 A MDA468 xenograft B 4000 LAPTM4b ) 3 3000 Control LAPTM4B

2000 actin

Tumor size (mm Tumor 1000

0 0102030405060 Days C D EF 80 200 60 Control LAPTM4B /p62 LC3 I 60 150 I I 40 LC3II /LC3 /LC3 II 40 100 II P62 20 20 50 actin Ratio of LC3 Ratio of LC3 0 Ratio of p62/actin 0 0 Ctrl L4B Ctrl L4B Ctrl L4B

Lysosomal transmembrane protein LAPTM4B promotes autophagy and tolerance to metabolic stress in cancer cells

Yang Li, Qing Zhang, Ruiyang Tian, et al.

Cancer Res Published OnlineFirst October 28, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-11-0940

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