EGFR-Mediated Beclin 1 Phosphorylation in Autophagy Suppression, Tumor Progression, and Tumor Chemoresistance

EGFR negatively regulates autophagy by binding to Beclin 1.
Active EGFR phosphorylates Beclin 1 and alters its interactome.
EGFR suppression of Beclin 1 may contribute to tumor progression in lung cancer.

Lung cancer responses to EGFR inhibitors may involve activation of Beclin 1. Image Credit: Cell Press

Summary
Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

Introduction
Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, links extracellular signals to cellular homeostasis. In normal cells, EGFR signaling is triggered by the binding of growth factors, such as epidermal growth factor (EGF), leading to homodimerization or heterodimerization with other EGFR family members (such as HER2/neu) and autophosphorylation of the intracellular domain (Lemmon and Schlessinger, 2010). The phosphotyrosines formed serve as a docking site for adaptor molecules, which results in the activation of signaling pathways including the Ras/MAPK pathway, the PI3K/Akt pathway, and STAT signaling pathways. In tumor cells, the tyrosine kinase activity of EGFR may be dysregulated by EGFR gene mutation, increased EGFR gene copy number, or EGFR protein overexpression, leading to aberrant EGFR signaling and increased tumor cell survival, proliferation, invasion, and metastasis ( Ciardiello and Tortora, 2008). EGFR signaling is deregulated in many human cancers, including those of the lung, head and neck, colon, pancreas, and brain.

The deregulation of EGFR in human cancers has led to the development of anticancer agents that target EGFR, including: (1) anti-EGFR antibodies that inhibit ligand binding and (2) small-molecule receptor tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, that block EGFR intracellular tyrosine kinase activity. Although the EGFR TKIs have shown limited clinical benefit in the majority of solid tumors, they are effective in non-small-cell lung carcinomas (NSCLCs) that harbor specific mutations in the tyrosine kinase domain of EGFR (most commonly, in-frame deletion in exon 19 around codons 746–750 or single-base substitution, L858R, in exon 21) (Ciardiello and Tortora, 2008, Lynch et al., 2004 and Pao and Chmielecki, 2010). Most patients with NSCLCs with EGFR mutations initially respond favorably to erlotinib or gefitinib, suggesting these mutations drive tumorigenesis. However, among tumors that initially respond to EGFR TKIs, most eventually acquire resistance, often due to the emergence of a secondary mutation, T790M, in the kinase domain of EGFR (Pao and Chmielecki, 2010).

Several studies have shown that EGFR signaling regulates autophagy, a lysosomal degradation pathway that functions in cellular homeostasis and protection against a variety of diseases, including cancer (Levine and Kroemer, 2008). The downstream targets of EGFR—PI3K, Akt, and mammalian target of rapamycin (mTOR)—are well-established negative regulators of autophagy (Botti et al., 2006). Moreover, EGFR inhibitors induce autophagy in NSCLCs (Gorzalczany et al., 2011 and Han et al., 2011) and other cancer cells (Fung et al., 2012). However, the links between EGFR signaling and autophagy remain poorly understood, particularly (1) the molecular mechanisms by which EGFR signaling suppresses autophagy, (2) the role of EGFR suppression of autophagy in lung cancer pathogenesis, and (3) the role of autophagy induction in the response to TKI therapy. EGFR inhibitor-induced autophagy in lung cancer cells has been postulated to exert either cytoprotective (Han et al., 2011) or cytotoxic (Gorzalczany et al., 2011) effects.

Conflicting results regarding the role of autophagy in the response or resistance to EGFR TKI treatment reflects broader uncertainties in the role of autophagy in cancer therapy (Rubinsztein et al., 2012). It is not understood in what contexts autophagy induction contributes to tumor progression or suppression and to tumor chemoresistance or chemosensitivity. There is a general consensus that autophagy prevents tumor initiation, as loss-of-function mutations of several different autophagy genes results in spontaneous tumorigenesis (beclin 1, Atg5, and Atg7) and/or increased chemical-induced tumorigenesis (Atg4C) in mice ( Rubinsztein et al., 2012). Despite this inhibitory role in tumor initiation, it has been proposed that autophagy may promote the growth of established tumors and contribute to chemoresistance, principally through its actions to prolong the survival of metabolically stressed neoplastic cells ( Rubinsztein et al., 2012).

To understand the relationship between oncogenic signaling, autophagy, and distinct stages of tumorigenesis, it is important to define the molecular mechanisms by which oncogenic signaling regulates autophagy. We recently showed that the oncogene Akt inhibits autophagy independently of mTOR signaling via serine phosphorylation of the essential autophagy protein, Beclin 1 (Wang et al., 2012), a haploinsufficient tumor suppressor protein frequently monoallelically deleted in human breast and ovarian cancer (Levine and Kroemer, 2008). Moreover, Akt-mediated phosphorylation of Beclin 1 contributes to Akt-dependent fibroblast transformation, supporting the concept that inactivation of Beclin 1-dependent autophagy plays a role in tumor initiation. However, it is not known whether oncogenic inactivation of Beclin 1 (or other autophagy proteins) influences progression of established tumors and/or their response to therapy.


Here, we identify the molecular basis by which EGFR tyrosine kinase activity regulates autophagy. We show that active EGFR binds to Beclin 1, leading to its tyrosine phosphorylation, alteration of its interactome, and inhibition of its autophagy function. A mutant of Beclin 1 containing phosphomimetic mutations in the EGFR-dependent tyrosine phosphorylation sites enhances autophagy suppression in EGFR-mutated NSCLC cells, resulting in enhanced tumor progression, altered tumor cell differentiation, and partial tumor resistance to EGFR TKI therapy. These findings demonstrate a heretofore unknown link between oncogenic receptor tyrosine kinases and the autophagy machinery, which may contribute to tumor progression and resistance to targeted therapy.

Source: Full Artical At - CELL PRESS

Drug combo supresses growth of late-stage prostate cancer turmors

By Natalie van Hoose

Low doses of metformin, a widely used diabetes medication, and a gene inhibitor known as BI2536 can successfully halt the growth of late-stage prostate cancer tumors, a Purdue University study finds.

Prostate cancer causes the second-highest number of cancer-related deaths in men in the U.S., and methods of treating advanced prostate cancer are limited.

Xiaoqi Liu (pronounced zhow-CHEE' LEE'-oo), associate professor of biochemistry and cancer research, and fellow researchers found that the drugs metformin and BI2536 can work together to suppress the spread of prostate cancer that resists all other available treatments, potentially prolonging patients' lives.

"We've found a promising way to treat late-stage prostate cancer," Liu said. "By combining low levels of two well-tolerated drugs, the progression of this disease could be significantly delayed. Completely curing the cancer at the advanced stage is pretty much impossible, but this treatment might manage it for a while - that's exciting."

A number of treatments exist for the earlier stages of prostate cancer, which grows slowly compared with many other cancers. Because prostate cancer cells need the male sex hormone androgen to develop, one way to treat the disease is to suppress androgen - a process known as castration. If the cancer continues to spread, the patient often undergoes chemotherapy. As a last resort, drugs that block the synthesis of androgen by prostate cancer cells can be used, but these medications only extend a patient's lifespan for several months.

New approaches to treating the most persistent forms of prostate cancer are "urgently needed," Liu said.

Adding to the challenge is the fact that castration treatment can inadvertently encourage the cancer to get tougher. It can heighten oxidative stress on the prostate gland, which increases the expression of Plk1, a gene that has been linked to many cancers. Over-expression of Plk1 can also trigger the synthesis of androgen.

"The goal of castration is to block androgen synthesis," Liu said. "But cancer cells eventually become 'smart' enough to make androgen anyhow, which is why the cancer continues to grow."

Additionally, castration can disrupt the body's metabolism and lead to insulin resistance, which also can stimulate the production of androgen. The cancer will spread until both of these side effects are stopped, Liu said.

Previous studies showed that metformin - an inexpensive, antidiabetic drug that has been commonly used for more than 40 years - is particularly potent to prostate cancer tumors.

Working with fellow researchers from Purdue, the University of Wisconsin-Madison and the Indiana University School of Medicine, Liu found that a combination of low levels of metformin and BI2536, a drug that stifles the activity of Plk1, could work in tandem to slow the growth of prostate tumors too advanced for current treatments by promoting the self-destruction of cancer cells and preventing androgen synthesis.

The drugs did not impact healthy prostate cells, a "key finding," Liu said. "Ideally, cancer therapy will have minimal effects on normal cells."

Because metformin helps regulate metabolism, it may reverse some of the metabolic damage caused by castration, he said.

The researchers tested the drugs in a classical cell culture assay of prostate cancer cells and in advanced prostate tumors in mice. Low concentrations of the drugs significantly slowed the development of cancer in both trials. The mice tumors were grown from the tumor cells of a late-stage prostate cancer patient, suggesting that the treatment would prove effective in humans.

"Those results were amazing," Liu said. "These are the first data we've generated from a real patient, so I was almost jumping in the air when I saw that it worked."

Liu said that the next step in the research is to test the combination of drugs in clinical trials. Further research is also needed to understand the underlying mechanism of metformin and why it is effective at suppressing prostate cancer

Source: Purdue Univesity