Cancer cells adapt FAM134B-BiP complex mediated ER-phagy to survive hypoxic stress

In a tumor microenvironment cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins in the endoplasmic reticulum (ER) which elicit unfolded protein response (UPR) as an adaptive mechanism. UPR activates autophagy enabling the degradation of misfolded/unfolded proteins. More recently, ER-specific autophagy has been implicated in the removal of damaged ER and restoration of ER-homeostasis. Our investigations reveal that during hypoxia induced ER-stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER-homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER-stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia activated proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. Our studies have further led to the identification of a pharmacological agent vitexin that disrupts FAM134B-BiP complex thereby inhibits ER-phagy and suppresses breast cancer progression in vivo.


Introduction 1
Cancers often encounter a characteristic microenvironment called tumor microenvironment 2 (TME) which comprises of a chemical microenvironment (pH, hypoxia, metabolite concentration) 3 and a cellular microenvironment (blood vessels, immune suppressor cells, fibroblasts, extracellular 4 matrix, stromal cells) which influences the growth of cancerous cells 1, 2, 3, 4 . Hypoxic environment 5 arises as a result of vascular insufficiency during the tumor expansion and progression 5 . It alters 6 the cancer cell metabolism and contributes to therapy resistance by activating certain adaptive 7 responses such as endoplasmic reticulum (ER) stress, anti-oxidative responses and autophagy 6 . 8 Therefore hypoxia is considered as a major impediment for effective cancer therapy 5, 7 . 9 ER is a multifunctional organelle, but it is central for protein synthesis, modifications and 10 transport. The disulphide bonds that are formed during protein synthesis are independent of oxygen 11 availability whereas, the bonds that are formed during the post-translational folding in the ER are 12 oxygen-dependent 8 . This process is altered during hypoxia resulting in the accumulation of 13 misfolded/unfolded proteins in the ER, perturbing its homeostasis. Thus, hypoxia directly impacts 14 protein modifications in the ER resulting in the activation of unfolded protein response (UPR) to 15 preserve ER homeostasis 9 . UPR is a signaling system which activates cellular responses 16 coordinated via three key regulators -inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase 17 (PERK) and activating transcription factor 6 (ATF6) 10,11,12,13 . Binding immunoglobulin protein 18 (BiP or glucose-regulatory protein 78 -Grp78) is a chaperone present abundantly in the ER which 19 transiently binds to the luminal domain of UPR receptors -IRE1, PERK and ATF6 14 . When the 20 misfolded/unfolded proteins begin to accumulate in the ER, BiP rapidly dissociates from the three 21 UPR signaling sensors and binds the exposed hydrophobic regions of the nascent polypeptides to 22 facilitate proper folding. In addition, UPR also induces autophagy as a key response to the stress 23 pathway activation in cancer cells which allows them to maintain metabolic homeostasis 15, 16, 17 . 24 Autophagy involves the sequestration of cytoplasmic components into autophagosomes which 25 then fuse with lysosomes and degrade their contents 18 . Although, autophagy is a constitutive 26 homeostatic mechanism which regulates intracellular recycling, it is also a major stress responsive 27 mechanism that facilitates the removal of damaged proteins and organelles 19 . Thus, autophagy 28 bestows tolerance to stress and sustains cell viability under hostile conditions and is considered a 29 "double-edged sword" because of its ability to suppress tumor yet promote tumor survival under 30 stress 19 . Despite accumulating evidences suggesting that autophagy is critical in cancer, it is still 31 a question of intense debate and remains complex 20 . For a long time autophagy was considered a 32 non-selective degradation pathway however, recent research has revealed that autophagy can 33 selectively degrade specific organelles including mitochondria (mitophagy), peroxisomes 34 (pexophagy), ER (ER-phagy or reticulophagy), nucleus (nucleophagy) 18 and aggregate-prone 35 proteins (aggrephagy) 21 . 36 ER-phagy was first described by Peter Walter's group where they demonstrated that selective 37 engulfment of ER into autophagosomes utilize several autophagy proteins induced by UPR and 38 these are essential for the survival of cells exposed to severe ER stress 22 . ER was originally 39 considered as the primary source of autophagosome membranes 23 and ER membranes observed 40 in autophagosomes was viewed as a result of bulk engulfment of cytosol 23,24

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Hypoxia is characterized by the stabilization of HIF-1a hence, we first investigated if HIF-1a is 59 expressed and stabilized in our model of MCF-7 breast cancer cells upon hypoxic stress induced 60 chemically using cobalt chloride (CoCl2) or growing cells in hypoxic environment (1% O2) for 61 24h. As expected, both chemical induction (CI) (Fig 1a) and hypoxic environment (HE) (Fig 1b) 62 resulted in HIF-1a expression and stabilization. Although CoCl2 has been widely used as a 63 chemical inducer of hypoxia, reports indicate that CoCl2 activates a complex relationship between 64 adaptive and cell death responses 36 . We observed HIF-1a expression and cell proliferation were 65 concentration dependent for CI-hypoxia (Ext Fig 1a). Time lapse imaging of MCF-7 cells treated 66 with CoCl2 at 500µM for 24h showed more than 2-fold increase in cell proliferation compared to analysis of UPR markers BiP, spliced XBP-1 (XBP-1s), CREB-2/ATF4 and CHOP showed 77 significant upregulation upon CI-hypoxia (Fig 1e). Increased expression of UPR target proteins 78 was also confirmed in cells grown in HE (Fig 1f). These data suggest that hypoxia induced HIF-79 1a and ER-stress response correlates with increased cancer cell proliferation.

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It is well-known that autophagy is required for the survival of hypoxic cancer cells 37 and it assists 81 in degrading misfolded/unfolded proteins to reestablish ER homeostasis 4, 38 . Hence, we next 82 investigated whether autophagy is induced in cancer cells under hypoxia. In cells subjected to CI-83 hypoxia ( Ext Fig 1f and 1h) and HE ( Ext Fig 1g and 1i), LC3B, a bonafide marker of autophagy 84 activation, showed increased conversion to its active form (LC3II) compared to the normoxic cells.

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Moreover, inhibition of lysosomal activity using concanamycin A lead to further increase in the 86 accumulation of LC3II in cells treated with CoCl2 (Fig 1g-1h). This was congruent with confocal  Hypoxia induces ER-phagy to maintain ER homeostasis 101 Various types of selective autophagy that are independent of p62 degradation have been described.

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In these processes, damaged organelles are selectively targeted into autophagosomes for 103 degradation. Since we observed that the ER is stressed during hypoxia (Fig 1d), we questioned if 104 the more-recently described ER-selective autophagy (ER-phagy) 40,41 was involved in the removal 105 of damaged ER. Microscopic analysis of cells expressing GFP-WIPI-1 and mCherry-ER-3, 106 showed increase in GFP-WIPI-1 puncta (autophagosomes) in cells subjected to hypoxia and they 107 were found to co-localize with mCherry-ER-3 (Fig 2a & Fig 2a). suggested that damaged ER is engulfed by autophagosomes to mitigate ER-stress caused by 112 hypoxia. However, during macroautophagy ER also provides membrane for autophagosome 113 8 formation 23, 24 . Hence, we next investigated the expression of ER-phagy-specific receptors such as 114 FAM134B, RTN3, Sec62, CCPG1 and the COPII subunit Sec24C which have been shown to target 115 ER for autophagosomal degradation. We did not observe any change in the expression of CCPG1, 116 RTN3 and Sec24C but a modest increase in Sec62 (Ext Fig 2b). In contrast, FAM134B decreased 117 consistently upon CI-hypoxia and in cells grown in HE (Fig 2c-2d). However, removal of hypoxic 118 stress by replacing CoCl2 with medium without CoCl2, restored the expression of FAM134B ( Fig   119   2e and Ext Fig 2c). We found that the decline in FAM134B was due to lysosomal degradation as 120 inhibition of lysosomal activity using concanamycin A prevented the degradation during HE 121 hypoxia (Fig 2f and Ext Fig 2d) and CI-hypoxia (Ext Fig 2e). We also observed a similar 122 degradation of FAM134B in U251 glioblastoma (Ext Fig 2f) and C32 melanoma (Ext Fig 2g) 123 indicating ER-phagy as a general mechanism in cancer cells to counteract hypoxia induced stress.  (Fig 2l). These observations indicate that hypoxia induces ER-phagy to overcome ER-135 9 stress and that FAM134B-dependent ER-phagy is vital for cancer cells to proliferate under hypoxic 136 stress.

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Hypoxia induced ER-phagy is BiP dependent 138 Since we deciphered that hypoxia leads to activation of UPR and FAM134B dependent ER-phagy, 139 we asked if this is HIF-1a dependent. Although depletion of HIF-1a downregulated hypoxia 140 induced UPR (Fig 3a-3e), degradation of FAM134B was not affected in cells subjected to CI-141 hypoxia (Fig 3f and 3h) and HE (Fig 3g and 3i). This suggests that hypoxia-induced ER-phagy 142 is independent of HIF-1a but, triggered by an alternative pathway most likely connected to the  Fig 3b). Strikingly, BiP co-immunoprecipitated with endogenous 150 FAM134B when MCF7 cells were subjected to hypoxia (Fig 3n). Taken together, these data show 151 that ER-phagy is a specific response to ER-stress and is coregulated by BiP and FAM134B.

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Above shown results revealed that ER-phagy alleviates ER-Stress response and facilitates the 154 survival and progression of hypoxic cells. Hence, we intended to pharmacologically target BiP 155 mediated ER-phagy to prevent cancer cell proliferation. For this, we have retrieved the high-156 resolution X-ray crystal structure of the protein from the Protein Data Bank (PDB ID: 5F0X.pdb, Resolution: 1.6 Å). Validation by Ramachandran Plot depicted that 98% and 2% of amino acids 158 are in favorable and allowed regions respectively (Ext Fig 4a). Therefore, we next performed 159 molecular docking studies using Schrödinger Suite 2015-3. A small set of small molecules library 160 was docked onto the binding site of the protein and identified an apigenin flavone glucoside, 161 vitexin as a potential molecule to target BiP. Docking scores are shown in Supplement Table 1.

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Vitexin showed lowest glide score towards BiP i.e., -8.3Kcal/mol (Supplement Table 1). The vitexin when compared to the protein without the binding of vitexin in the binding site (Fig. 4e). 182 Based on these findings using molecular docking and molecular dynamics approaches, we 183 subjected vitexin for further in-vitro validation.

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Vitexin prevents FAM134B-BiP interaction and inhibits ER-phagy 185 Since our in silico molecular dynamics studies deciphered vitexin as a potential inhibitor of BiP, 186 we next examined the expression of BiP in hypoxic cells treated with vitexin. Consistently, vitexin 187 treatment downregulated BiP expression during hypoxia (Fig 5a-5b). Indeed, treatment with 188 vitexin also downregulated CI-hypoxia induced UPR at the mRNA (Ext Fig 5a-5d) and protein 189 levels (Ext Fig 5e). Next, we asked if treatment with vitexin prevented the interaction of 190 FAM134B with BiP during hypoxia. Strikingly, BiP did not coimmunoprecipitate with FAM134B 191 upon vitexin treatment in hypoxic cells (Fig 5c). Furthermore, immunoblot analysis revealed that 192 vitexin prevents autophagosomal degradation of FAM134B during CI-hypoxia (Fig 5d and 5f) 193 and HE-hypoxia (Fig 5e and 5g).  Having shown that vitexin inhibits ER-phagy we surmised that it could inhibit cancer cell 204 proliferation under hypoxic stress. As expected, vitexin treatment effectively prevented the 205 increase in cell proliferation upon hypoxic stimuli (Fig 6a). As we propose that ER-phagy resolves 206 ER-stress, we also explored if vitexin can synergistically inhibit cancer cell growth with an ER-207 stress inducer tunicamycin. We observed that vitexin and tunicamycin synergistically inhibited 208 cancer cell growth (Fig 6b). We next asked if vitexin can reduce tumor burden in female balb/c 209 athymic (nuþ/nuþ) mice xenografted with MCF7 cells. We found that after 13, 17 and 21 days of 210 vitexin treatment, tumor volume was significantly reduced compared to the vehicle treated mice 211 (Fig 6c-6d). Taken together, we could conclude that vitexin shows a higher therapeutic potential 212 in treating breast cancer.

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Identification of receptors that specifically target damaged organelles and proteins into 215 autophagosomes for degradation assists in discriminating from functional organelles. More 216 recently, specific receptors have been identified to target damaged or excess parts of ER into 217 autophagosomes for elimination and this process has been termed as ER-phagy. Thus far, this 218 process has not been directly implicated in disease pathologies. Here, we report that cancer cells 219 undergo ER-phagy regulated by FAM134B-BiP complex when they are subjected to hypoxic 220 stress which helps the cells to mitigate ER-stress and promote cell proliferation (Fig 6e). 221 Numerous studies have confirmed that the TME promotes cancer progression due to the ability of organelles to maintain cellular homeostasis. In line with this, hypoxia has been shown to target 247 mitochondria (mitophagy) 50 and peroxisomes (pexophagy) 51 for autophagic degradation.

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As hypoxia causes damage to ER, a failure to restore ER structure and homeostasis could be lethal 249 to cells. Restoration of ER is achieved by the removal of excess and damaged ER caused by ER-250 stress. Recent evidences suggest that ER can be selectively targeted for lysosomal degradation by 251 ER-phagy 52 . UPR has also been linked to the activation of ER-phagy however, no direct evidences 252 linking hypoxia and ER-phagy has been reported thus far. Our data convincingly show that ER is stimulated ER-stress reduces the viability of FAM134B-depleted breast cancer cells. We also 265 observed a modest increase in Sec62 which has been shown to specifically regulate the recovery 266 of ER 28, 53 but was not targeted for autophagic degradation during hypoxia. Notably, hypoxia 267 upregulates FAM134B expression in chronic myeloid leukemia (CML) cells and is correlated with 268 pro-survival 54 . It is also speculated that its upregulation is HIF-1a dependent 55 however, silencing 269 of HIF-1a did not alter the relative expression of FAM134B during hypoxia compared to normoxia. indicates that it abrogates the ability of cancer cells to overcome ER-stress. It also suggests that 292 vitexin not only prevents BiP from binding to FAM134B but also prevents the ability of BiP to 293 chaperone unfolded/misfolded proteins. Furthermore, induction of ER-stress with tunicamycin 294 combined with ER-phagy inhibition using vitexin synergistically stunted cell proliferation.

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Therefore, we surmise that unresolved ER-stress is detrimental to cell survival and proliferation.

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Disruption of ER and ER homeostasis could lead to the death of cells and cause disease 297 pathologies. However, during the late stages of cancer when cancer cells are under various 298 metabolic stresses including hypoxia, they adapt mechanisms such as ER-selective autophagy to 299 overcome the damage to ER and the associated cellular processes. Therefore, targeting such 300 adaptive mechanisms is a potential way forward to treat cancer. Our data reported here unveils 301 FAM134B-BiP complex-mediated ER-phagy as a novel mechanism by which cancer cells prevail 302 over hypoxia induced proteotoxic stress and targeting ER-phagy machinery as a prospective 303 therapeutic strategy to treat cancer.    Table 1.   The next day, following treatment with CoCl2 and vitexin cells were set up for time-lapse imaging 371 20 in the Leica SP8 confocal or Leica THUNDER imager over 24 h duration at an interval of 20min.

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The data was processed and analyzed using imageJ software.