Schroeder, Barbara

Abstract Hepatocellular endocytosis is a highly dynamic process responsible for the internalization of a variety of different receptor ligand complexes, trophic factors, lipids, and, unfortunately, many different pathogens. The uptake of these external agents has profound effects on seminal cellular processes including signaling cascades, migration, growth, and proliferation. The hepatocyte, like other well‐polarized epithelial cells, possesses a host of different endocytic mechanisms and entry routes to ensure the selective internalization of cargo molecules. These pathways include receptor‐mediated endocytosis, lipid raft associated endocytosis, caveolae, or fluid‐phase uptake, although there are likely many others. Understanding and defining the regulatory mechanisms underlying these distinct entry routes, sorting and vesicle formation, as well as the postendocytic trafficking pathways is of high importance especially in the liver, as their mis‐regulation can contribute to aberrant liver pathology and liver diseases. Further, these processes can be “hijacked” by a variety of different infectious agents and viruses. This review provides an overview of common components of the endocytic and postendocytic trafficking pathways utilized by hepatocytes. It will also discuss in more detail how these general themes apply to liver‐specific processes including iron homeostasis, HBV infection, and even hepatic steatosis. © 2014 American Physiological Society. Compr Physiol 4:1403‐1417, 2014.

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 Communications2017;1:140‐152)

Abstract 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

Abstract The HER2 receptor is over-expressed in about 25% of breast carcinomas and correlates with more aggressive breast cancer disease, poor prognosis as well as resistance to chemo- and endocrine therapies. The strong correlation between HER2 levels/activity and the malignancy of the disease made it a preferred target for anti-cancer therapy. For example, Trastuzumab, which targets HER2 homo-dimerization and Pertuzumab, which interferes with HER2 hetero-dimerization, are in clinical use. Unfortunately, Trastuzumab monotherapy is only partially effective and the majority of breast cancer patients who initially respond to Trastuzumab rapidly develop resistance to the drug. Thus, novel strategies/agents are in need, especially drugs that prevent hetero-dimerization with other HER family members. Structural analyses revealed that sub-domain III in the extracellular domain (ECD) of the HER family members is responsible for dimerization. Therefore, we hypothesized that disabling the dimerization loop in the HER2-ECD sub-domain III would ultimately convert HER2 to a non-functional receptor. Therefore, we introduced a series of deletions in the HER2-ECD sub-domain III and determined their oncogenic properties compared to HER2wt expressing 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, both in the context of HER2 over-expression and ligand-induced trans-activation of HER2. Expression of the HER2Δ6 variant failed to promote anchorage-independent growth and interfered with the activation/Tyr phosphorylation of HER1, HER2 and HER3. Moreover, this mutant failed to promote resistance to Paclitaxel treatment in HER2-overexpressing breast cancer cells. 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), most likely due to a intracellular trafficking defect that mistargets the receptor to or traps it in an endomembrane compartment. Compared to HER2wt-expressing cells, the HER2Δ6 mutant proves to be even more effective in inhibiting the oncogenic properties of the receptor than the current drugs of choice such as Trastuzumab, Pertuzumab and Cetuximab alone or in combination as measured by anchorage-independent growth. These findings reveal that the HER2-ECD bears an essential "activating" region that is indispensable for HER2 homo-and heterodimerization. Elimination of this "activating" element in HER2 seems to recapitulate and greatly improve the combined actions of Trastuzumab and Pertuzumab. These findings offer a strong rationale for developing this peptide sequence into a valuable anti-HER2 therapeutic drug. Citation Format: Barbara Schroeder, Menendez Javier, Espinoza Ingrid, Lupu Ruth. Blocking a key region in the HER2 subdomain III inhibits the HER2-network in patients with resistance to HER2-targeted therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-08.

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. (H epatology 2015;61:1896–1907)

Lipid droplets (LDs) are lipid storage organelles that in hepatocytes may be catabolized by autophagy for use as an energy source, but the membrane-trafficking machinery regulating such a process is poorly characterized. We hypothesized that the large GTPase Dynamin 2 (Dyn2), well known for its involvement in membrane deformation and cellular protein trafficking, could orchestrate autophagy-mediated LD breakdown. Accordingly, depletion or pharmacologic inhibition of Dyn2 led to a substantial accumulation of LDs in hepatocytes. Strikingly, the targeted disruption of Dyn2 induced a dramatic four- to fivefold increase in the size of autolysosomes. Chronic or acute Dyn2 inhibition combined with nutrient deprivation stimulated the excessive tubulation of these autolysosomal compartments. Importantly, Dyn2 associated with these tubules along their length, and the tubules vesiculated and fragmented in the presence of functional Dyn2. These findings provide new evidence for the participation of the autolysosome in LD metabolism and demonstrate a novel role for dynamin in the function and maturation of an autophagic compartment.

Ubiquitination of the epidermal growth factor receptor (EGFR) by cbl and its cognate adaptor cbl-interacting protein of 85 kDa (CIN85) is known to play an essential role in directing this receptor to the lysosome for degradation. The mechanisms by which this ubiquitin modification is regulated are not fully defined, nor is it clear where this process occurs. In this study we show that EGFR activation leads to a pronounced src-mediated tyrosine phosphorylation of CIN85 that subsequently influences EGFR ubiquitination. Of importance, phospho-CIN85 interacts with the Rab5-positive endosome, where it mediates the sequestration of the ubiquitinated receptor into multivesicular bodies (MVBs) for subsequent degradation. These findings provide novel insights into how src- kinase–based regulation of a cbl adaptor regulates the fate of the EGFR.