Barbara Schroeder

The small GTPase Rab7 as a central regulator of hepatocellular lipophagy

Autophagy is a central mechanism by which hepatocytes catabolize lipid droplets (LDs). Currently, the regulatory mechanisms that control this important process are poorly defined. The small guanosine triphosphatase (GTPase) Rab7 has been implicated in the late endocytic pathway and is known to associate with LDs, although its role in LD breakdown has not been tested. In this study, we demonstrate that Rab7 is indispensable for LD breakdown (“lipophagy”) in hepatocytes subjected to nutrient deprivation. Importantly, Rab7 is dramatically activated in cells placed under nutrient stress; this activation is required for the trafficking of both multivesicular bodies and lysosomes to the LD surface during lipophagy, resulting in the formation of a lipophagic “synapse.” Depletion of Rab7 leads to gross morphological changes of multivesicular bodies, lysosomes, and autophagosomes, consequently leading to attenuation of hepatocellular lipophagy. Conclusion: These findings provide additional support for the role of autophagy in hepatocellular LD catabolism while implicating the small GTPase Rab7 as a key regulatory component of this essential process. (Hepatology 2015;61:1896–1907)

Lipid droplet breakdown requires dynamin 2 for vesiculation of autolysosomal tubules in hepatocytes.

Dynamin 2 is required for starvation-mediated breakdown of lipid droplets in hepatocytes by promoting vesiculation of autolysosomal tubules to release protolysosomes.

A Dyn2-CIN85 complex mediates degradative traffic of the EGFR by regulation of late endosomal budding.

The epidermal growth factor receptor (EGFR) is over‐expressed in a variety of human cancers. Downstream signalling of this receptor is tightly regulated both spatially and temporally by controlling its internalization and subsequent degradation. Internalization of the EGFR requires dynamin 2 (Dyn2), a large GTPase that deforms lipid bilayers, leading to vesicle scission. The adaptor protein CIN85 (cbl‐interacting protein of 85 kDa), which has been proposed to indirectly link the EGFR to the endocytic machinery at the plasma membrane, is also thought to be involved in receptor internalization. Here, we report a novel and direct interaction between Dyn2 and CIN85 that is induced by EGFR stimulation and, most surprisingly, occurs late in the endocytic process. Importantly, disruption of the CIN85–Dyn2 interaction results in accumulation of internalized EGFR in late endosomes that become aberrantly elongated into distended tubules. Consistent with the accumulation of this receptor is a sustention of downstream signalling cascades. These findings provide novel insights into a previously unknown protein complex that can regulate EGFR traffic at very late stages of the endocytic pathway.

Vav1 as a Central Regulator of Invadopodia Assembly

Invadopodia are protrusive structures used by tumor cells for degradation of the extracellular matrix to promote invasion [1]. Invadopodia formation and function are regulated by cytoskeletal-remodeling pathways and the oncogenic kinase Src. The guanine nucleotide exchange factor Vav1, which is an activator of Rho family GTPases, is ectopically expressed in many pancreatic cancers, where it promotes tumor cell survival and migration [2, 3]. We have now determined that Vav1 is also a potent regulator of matrix degradation by pancreatic tumor cells as depletion of Vav1 by siRNA-mediated knockdown inhibits the formation of invadopodia. This requires the exchange function of Vav1 toward the GTPase Cdc42, which is required for invadopodia assembly [4, 5]. In addition, we have determined that Src-mediated phosphorylation and activation of Vav1 are both required for, and, unexpectedly, sufficient for, invadopodia formation. Expression of Vav1 Y174F, which mimics its activated state, is a potent inducer of invadopodia formation through Cdc42, even in the absence of Src activation and phosphorylation of other Src substrates, such as cortactin. Thus, these data identify a novel mechanism by which Vav1 can enhance the tumorigenicity and invasive potential of cancer cells. These data suggest that Vav1 promotes the matrix-degrading processes underlying tumor cell migration and further, under conditions of ectopic Vav1 expression, that Vav1 is a central regulator and major driver of invasive matrix remodeling by pancreatic tumor cells.

Suppression of endogenous lipogenesis induces reversion of the malignant phenotype and normalized differentiation in breast cancer

The correction of specific signaling defects can reverse the oncogenic phenotype of tumor cells by acting in a dominant manner over the cancer genome. Unfortunately, there have been very few successful attempts at identifying the primary cues that could redirect malignant tissues to a normal phenotype. Here we show that suppression of the lipogenic enzyme fatty acid synthase (FASN) leads to stable reversion of the malignant phenotype and normalizes differentiation in a model of breast cancer (BC) progression. FASN knockdown dramatically reduced tumorigenicity of BC cells and restored tissue architecture, which was reminiscent of normal ductal-like structures in the mammary gland. Loss of FASN signaling was sufficient to direct tumors to a reversed phenotype that was near normal when considering the development of polarized growth-arrested acinar-like structure similar to those formed by nonmalignant breast cells in a 3D reconstituted basement membrane in vitro. This process, in vivo, resulted in a low proliferation index, mesenchymal-epithelial transition, and shut-off of the angiogenic switch in FASN-depleted BC cells orthotopically implanted into mammary fat pads. The role of FASN as a negative regulator of correct breast tissue architecture and terminal epithelial cell differentiation was dominant over the malignant phenotype of tumor cells possessing multiple cancer-driving genetic lesions as it remained stable during the course of serial in vivo passage of orthotopic tumor-derived cells. Transient knockdown of FASN suppressed hallmark structural and cytosolic/secretive proteins (vimentin, N-cadherin, fibronectin) in a model of EMT-induced cancer stem cells (CSC). Indirect pharmacological inhibition of FASN promoted a phenotypic switch from basal- to luminal-like tumorsphere architectures with reduced intrasphere heterogeneity. The fact that sole correction of exacerbated lipogenesis can stably reprogram cancer cells back to normal-like tissue architectures might open a new avenue to chronically restrain BC progression by using FASN-based differentiation therapies.

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase.

Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments.

Ethanol exposure inhibits hepatocyte lipophagy by inactivating the small guanosine triphosphatase Rab7

Alcohol consumption is a well‐established risk factor for the onset and progression of fatty liver disease. An estimated 90% of heavy drinkers are thought to develop significant liver steatosis. For these reasons, an increased understanding of the molecular basis for alcohol‐induced hepatic steatosis is important. It has become clear that autophagy, a catabolic process of intracellular degradation and recycling, plays a key role in hepatic lipid metabolism. We have shown that Rab7, a small guanosine triphosphatase known to regulate membrane trafficking, acts as a key orchestrator of hepatocellular lipophagy, a selective form of autophagy in which lipid droplets (LDs) are specifically targeted for turnover by the autophagic machinery. Nutrient starvation results in Rab7 activation on the surface of the LD and lysosomal compartments, resulting in the mobilization of triglycerides stored within the LDs for energy production. Here, we examine whether the steatotic effects of alcohol exposure are a result of perturbations to the Rab7‐mediated lipophagic pathway. Rats chronically fed an ethanol‐containing diet accumulated significantly higher levels of fat in their hepatocytes. Interestingly, hepatocytes isolated from these ethanol‐fed rats contained juxtanuclear lysosomes that exhibited impaired motility. These changes are similar to those we observed in Rab7‐depleted hepatocytes. Consistent with these defects in the lysosomal compartment, we observed a marked 80% reduction in Rab7 activity in cultured hepatocytes as well as a complete block in starvation‐induced Rab7 activation in primary hepatocytes isolated from chronic ethanol‐fed animals. Conclusion: A mechanism is supported whereby ethanol exposure inhibits Rab7 activity, resulting in the impaired transport, targeting, and fusion of the autophagic machinery with LDs, leading to an accumulation of hepatocellular lipids and hepatic steatosis. (Hepatology Communications 2017;1:140‐152)

Abstract 133: Identification of a key region in the HER2 subdomain III required for transformation capability

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA About 25% of breast carcinomas are characterized by over-expression and/or amplification of the HER2 receptor, one of four members of the HER family of receptor tyrosine kinases. The strong correlation between HER2 levels/activity with more aggressive breast cancer disease, poor prognosis as well as resistance to chemo- and endocrine therapies made it a preferred target for anti-cancer therapy. However, current treatment strategies are only partially effective and the majority of breast cancer patients who initially respond favorably rapidly develop resistance to treatments for still not fully understood reasons. Thus, novel strategies/agents are in need. Structural analyses revealed that sub-domain III in the extracellular domain (ECD) of the HER family members is responsible for dimerization. We hypothesized that disrupting the dimerization loop in the HER2-ECD sub-domain III would ultimately convert HER2 to a non-functional receptor with diminished transforming capacity. To this end, we generated a series of HER2-ECD deletion mutants located in sub-domain III and assessed their oncogenic potential compared to the HER2-wild type (WT) after their expression in MCF10A cells. Importantly, a small deletion (16 amino acids) of the HER2 extracellular domain (named HER2Δ6) abolished its homo- and hetero-dimerization and profoundly affected HER2-catalyzed activation of the HER network. Moreover, this mutant was not able to transform MCF10 cells as it failed to promote anchorage-independent growth and interfered with the activation/Tyr phosphorylation of HER1, HER2 and HER3. Compared to HER2-WT-expressing cells, the HER2α6 mutant proved to be even more effective in inhibiting the oncogenic properties of the receptor than the current drugs of choice such as Trastuzumab and Pertuzumab alone or in combination. In addition, while expression of HER2-WT conferred resistance of MCF10A cells to Paclitaxel, the HER2Δ6 variant failed to do so. To determine the molecular mechanisms underlying this behavior, we assessed the mutant and WT expressing cells lines morphologically and biochemically and demonstrated that the HER2Δ6 is absent from the plasma membrane (PM) in MCF10A cells. Interestingly, we found that the mutant receptor displayed an intracellular trafficking defect and was trapped in the endoplasmic reticulum (ER). Our results reveal that the HER2-ECD bears an essential “activating” region that is indispensable for HER2-mediated oncogenic transformation. Targeting and eliminating this “activating” element in HER2 seems to provide a strong innovative approach for developing a valuable novel anti-HER2 therapeutic drug that would specifically benefit patients with HER2 positive tumors resistant to current therapies. Citation Format: Barbara Schroeder, Ghiara Lugo, Javier Menendez, Ingrid Espinoza, Ruth Lupu. Identification of a key region in the HER2 subdomain III required for transformation capability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 133. doi:10.1158/1538-7445.AM2015-133