Decoding the growth advantage of hypoxia-sensitive lung cancer.

Exquisite control of responses to changes in ambient oxygen concentrations is a basic need for the survival of all organisms. Through evolution, a complex system has emerged for rapid responses to acute hypoxic changes. Interestingly, highly malignant, rapidly proliferative cancers including adenocarcinomas also exploit the native mechanistic arsenal of cells to adapt to tissue hypoxia, especially within central regions of the tumor. Hypoxia promotes tumor invasiveness and metastasis, a process largely accomplished by a program organized by the stabilization of hypoxia-inducible factors (HIFs), which function as indispensable transcription factors that bind to hypoxic response elements within a plethora of target genes to induce metabolic changes (1). HIF-1 is a heterodimer consisting of HIF-1α and HIF-1β, which are both highly conserved and present widely among mammalian cell types. HIF-1β is ubiquitously expressed, whereas HIF-1α exhibits low-level expression under normoxic conditions, due to its rapid turnover through ubiquitin proteasomal degradation. However, HIF-1α is induced during hypoxia. High levels of HIF-1 are observed in a broad range of human cancers due to the tumor hypoxic microenvironment, and activation of HIF-1α is associated with a poor prognosis in both small cell and non–small cell lung cancers (2, 3). HIF-1α activation induces the transcription of target genes associated with angiogenesis, proliferation, chemotherapy and radiation resistance, metastasis, genetic instability, immune evasion, and metabolism. In this issue of the Journal, Queisser and colleagues (pp. 688–698) provide evidence that HIF proteins are involved in eliminating a tumor suppressor protein, protein kinase Cζ (PKCζ), in lung adenocarcinoma (4). Hence, HIF represents a potentially attractive therapeutic target in cancer therapy. Approaches to suppress HIF-1 signaling have primarily concentrated on depleting cellular HIF-1α protein levels through inhibiting its gene transcription or facilitating its degradation, or blocking the formation of HIF-1 transcription factor complexes, which, in turn, inhibit HIF-1 target gene expression (5). Several preclinical and clinical agents have been identified that inhibit HIF-1 activity, thereby contributing to their antitumor efficacy (6). However, as with many small molecule antagonists, a major limitation using HIF antagonists may reside in the potential for off-target effects, especially given the vast array of genes regulated by these proteins. A more selective means perhaps to disrupt HIF signaling as it relates to tumorigenesis is to target the gene product downstream that is induced by HIFs. Queisser and colleagues show that HIFs trigger an increase in the levels of a ubiquitin E3 ligase component, termed heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1), that in turn mediates the elimination of PKCζ from cells. E3 ligases are members of the ubiquitin proteasome system (UPS), an elaborate program that orchestrates the selective disposal of ∼80% of cellular proteins. Its proximal activity is modulated through the activity of specific enzymes (E1 activating, E2 conjugating, and E3 ligases). There are hundreds of ubiquitin E3 ligases in cells, each relatively specific for a particular target (7). E3 ligases working in concert with E2 conjugating enzymes covalently attach a ubiquitin moiety to a lysine residue within a protein. When four or more ubiquitins are ligated to a substrate, the protein is polyubiquitinated and often targeted to the 26S proteasome, a structure where proteins are efficiently degraded within the cell. The ubiquitin moiety itself harbors seven lysine residues, and therefore can be ligated to incoming ubiquitins forming varying branching patterns of polyubiquitinated proteins. For example, lysine 48 (K48)-linked ubiquitin chains brand substrates for degradation by the 26S proteasome. Relatively recently, it was demonstrated that a ubiquitin E3 ligase complex, termed linear ubiquitin chain assembly complex (LUBAC), was shown to catalyze linear ubiquitination by covalently attaching an incoming ubiquitin to the amino-terminal methionine (M1) residue of the preceding ubiquitin molecule (8). LUBAC and M1–ubiquitin linkages act as crucial components regulating inflammation and immune responses, as M1 ubiquitination is required in tumor necrosis factor–induced nuclear factor-κB signaling (9). LUBAC is a multiprotein complex consisting of HOIL-1L, HOIL-1–interacting protein (HOIP), and Shank-associated RH domain–interacting protein (SHARPIN). The LUBAC component HOIL-1L was also reported to bind PKCζ (10), and in some proteins forms K48 polyubiquitin chains for degradation by the 26S proteasome (11, 12). Queisser and colleagues observed that patients with lung adenocarcinoma and glioblastoma multiforme display high levels of HOIL-1L and low levels of the tumor suppressor protein PKCζ (4). Moreover, the authors discovered the mechanistic basis for this observation whereby in adenocarcinoma cells a rapid adaptation response to hypoxia occurs involving activation of HIFs that increase the expression of HOIL-1L (Figure 1). This E3 ligase component was sufficient to form K48– ubiquitin chains in PKCζ, targeting it for proteasomal degradation. The authors also showed in mouse tumor xenograft studies that depletion of HOIL-1 resulted in a lower tumor burden in nude (immunodeficient) mice implanted with adenocarcinomas. The results are important in that they are the first demonstration that a hypoxia-sensitive ubiquitin E3 ligase targets a tumor suppressor. The data provide an alternative conceptual model perhaps justifying molecular strategies targeting HOIL-1L using small molecule antagonists or interrupting the association between HOIL-1L and PKCζ (4). This approach may provide greater selectively versus therapeutic targeting of more promiscuous factors that regulate transcriptional networks. Figure 1. A hypoxia-driven pathway for lung cancer. Under normal conditions, E3 ubiquitin ligase von Hippel-Lindau (VHL) labels hypoxia-inducible factor-α (HIF-α) for polyubiquitination and proteasomal degradation in the 26S proteasome. During hypoxia, . Pharmacologic agents targeting the UPS represent a new frontier in cancer therapy (13). Bortezomib, a nonselective 20S proteasome inhibitor, is the first agent available for human use that has shown success in hematologic malignancies including multiple myeloma (MM). This agent is now U.S. Food and Drug Administration approved for mantle cell lymphoma, and newer-generation 20S proteasome inhibitors are now Food and Drug Administration approved for relapsed or refractory MM (14). However, bortezomib has a dose-limiting side effect of peripheral neuropathy, and efforts are underway in the preclinical development of newer agents that inhibit steps upstream of the proteasome, including E3 ligases. For instance, BC-1215, a small molecule inhibitor of the E3 ligase subunit, Fbxo3, was recently synthesized to block the degradation of another E3 subunit protein, Fbxl2 (15). This latter protein mediates degradation of some cell cycle regulators. Thus, BC-1215 or related congeners may be useful for study in neoplasia. The elucidation of E3 ligase–substrate pairs as shown by Queisser and colleagues provides an excellent example where a mechanistic model decodes phenotypic observations in human disease that may present an opportunity for future pharmacologic intervention.