Beclin 1 is a haplo-insufficient tumor suppressor that is decreased in many human tumors. The function of Beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, Beclin 1 is a core component of the Vps34/Class III PI3K (PI3KC3) and Vps15/p150 complex that regulates multiple membrane trafficking events. In the current study, we describe an alternative mechanism of action for Beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by Beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P-) endosomes to PI3P+ endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P-/APPL+ signaling competent compartment. As a result, suppression of BECN1 sustains growth factor stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, Beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for Beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of Beclin 1 expression would enhance breast cancer progression.

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◥Beclin 1 has nonautophagic functions that include its ability to regulate endocytic receptor trafficking. However, the contribution of this function to tumor suppression is poorly understood. Here, we provide in vivo evidence that Beclin 1 suppresses tumor proliferation by regulating the endocytic trafficking and degradation of the EGFR and transferrin (TFR1) receptors. Beclin 1 promoted endosomal recruitment of hepatocyte growth factor tyrosine kinase substrate (HRS), which was necessary for sorting surface receptors to intraluminal vesicles for signal silencing and lysosomal degra-dation. In tumors with low Beclin 1 expression, endosomal HRS recruitment was diminished and receptor function was sustained. Collectively, our results demonstrate a novel role for Beclin 1 in impeding tumor growth by coordinating the regulation of key growth factor and nutrient receptors. These data provide an explanation for how low levels of Beclin 1 facilitate tumor proliferation and contribute to poor cancer outcomes.Significance: Beclin 1 controls the trafficking fate of growth regulatory receptors to suppress tumor proliferation.

The insulin receptor substrate (IRS) proteins serve as essential signaling intermediates for the activation of PI3K by both the insulin-like growth factor 1 receptor (IGF-1R) and its close family member, the insulin receptor (IR). Although IRS-1 and IRS-2 share significant homology, they regulate distinct cellular responses downstream of these receptors and play divergent roles in breast cancer. To investigate the mechanism by which signaling through IRS-1 and IRS-2 results in differential outcomes, we assessed the involvement of the microtubule cytoskeleton in IRS-dependent signaling. Treatment with drugs that either stabilize or disrupt microtubules reveal that an intact microtubule cytoskeleton contributes to IRS-2-but not IRS-1-mediated activation of AKT by IGF-1. Proximal IGF-1R signaling events, including IRS tyrosine phosphorylation and recruitment of PI3K, are not inhibited by microtubule disruption, indicating that IRS-2 requires the microtubule cytoskeleton at the level of downstream effector activation. IRS-2 colocalization with tubulin is enhanced upon Taxol-mediated microtubule stabilization, which, together with the signaling data, suggests that the microtubule cytoskeleton may facilitate access of IRS-2 to downstream effectors such as AKT. Of clinical relevance is that our data reveal that expression of IRS-2 sensitizes breast carcinoma cells to apoptosis in response to treatment with microtubule-disrupting drugs, identifying IRS-2 as a potential biomarker for the response of breast cancer patients to Vinca alkaloid drug treatment.Insulin receptor substrate 1 (IRS-1) 2 and IRS-2 are cytoplasmic adaptors for the insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R), and they play a major role in determining the cellular response to stimulation of these receptors (1). Notably, the IRS proteins are required for the activation of PI3K downstream of the IR and IGF-1R, which activate AKT and mechanistic target of rapamycin (mTOR) to promote proliferation, survival, motility, protein synthesis, and glucose metabolism (2-5). IRS-1 and IRS-2 are expressed ubiquitously in humans, including in the normal and malignant mammary epithelium (1). Despite their considerable sequence homology, IRS-1 and IRS-2 play divergent roles in breast cancer. In vitro, studies to assess IGF-1-dependent signaling through the IRS proteins in breast carcinoma cells have revealed that IRS-1 primarily regulates proliferation and survival, whereas IRS-2 regulates motility, invasion, and glycolysis (6 -10). In vivo, overexpression of either IRS-1 or IRS-2 in the mouse mammary gland promotes mammary tumorigenesis (11). However, metastasis is diminished in the absence of Irs-2 expression and increased in the absence of Irs-1 expression (9, 12).Differential localization patterns of IRS-1 and IRS-2 in human breast tumors suggest one explanation for their divergent functions in breast cancer (13). In normal breast tissue, ductal carcinoma in situ, and invasive breast tumors, IRS-1 is primarily localized in the nucleus a...

Although the insulin receptor substrate (IRS) proteins IRS1 and IRS2 share considerable homology and activate common signaling pathways, their contributions to breast cancer are distinct. IRS1 has been implicated in the proliferation and survival of breast tumor cells. In contrast, IRS2 facilitates glycolysis, invasion, and metastasis. To determine the mechanistic basis for IRS2-dependent functions, we investigated unique structural features of IRS2 that are required for invasion. Our studies revealed that the ability of IRS2 to promote invasion is dependent upon upstream insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (IR) activation and the recruitment and activation of phosphatidylinositol 3-kinase (PI3K), functions shared with IRS1. In addition, a 174-amino-acid region in the IRS2 C-terminal tail, which is not conserved in IRS1, is also required for IRS2-mediated invasion. Importantly, this "invasion (INV) region" is sufficient to confer invasion-promoting ability when swapped into IRS1. However, the INV region is not required for the IRS2-dependent regulation of glucose uptake. Bone morphogenetic protein 2-inducible kinase (BMP2K) binds to the INV region and contributes to IRS2-dependent invasion. Taken together, our data advance the mechanistic understanding of how IRS2 regulates invasion and reveal that IRS2 functions important for cancer can be independently targeted without interfering with the metabolic activities of this adaptor protein.