Byron Hann

Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3

Oncogenic tyrosine kinases have proved to be promising targets for the development of highly effective anticancer drugs. However, tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (HER) family show only limited activity against HER2-driven breast cancers, despite effective inhibition of epidermal growth factor receptor (EGFR) and HER2 in vivo. The reasons for this are unclear. Signalling in trans is a key feature of this multimember family and the critically important phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 (refs 9, 10). Here we show that HER3 and consequently PI(3)K/Akt signalling evade inhibition by current HER-family TKIs in vitro and in tumours in vivo. This is due to a compensatory shift in the HER3 phosphorylation–dephosphorylation equilibrium, driven by increased membrane HER3 expression driving the phosphorylation reaction and by reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt-mediated negative-feedback signalling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abrogation of HER3 resistance by small interfering RNA knockdown restores potent pro-apoptotic activity to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumours and the therapeutic promise of this oncoprotein target. However, because HER3 signalling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to silence oncogenic HER2 signalling effectively. The biologic marker with which to assess the efficacy of HER TKIs should be the transphosphorylation of HER3 rather than autophosphorylation.

A Mutant-p53/Smad Complex Opposes p63 to Empower TGFβ-Induced Metastasis

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

The signal recognition particle in S. cerevisiae

We have identified the Saccharomyces cerevisiae homolog of the signal recognition particle (SRP) and characterized its function in vivo. S. cerevisiae SRP is a 16S particle that includes a homolog of the signal sequence-binding protein subunit of SRP (SRP54p) and a small cytoplasmic RNA (scR1). Surprisingly, the genes encoding scR1 and SRP54p are not essential for growth, though SRP-deficient cells grow poorly, suggesting that SRP function can be partially by-passed in vivo. Protein translocation across the ER membrane is impaired in SRP-deficient cells, indicating that yeast SRP, like its mammalian counterpart, functions in this process. Unexpectedly, the degree of the translocation defect varies for different proteins. The ability of some proteins to be efficiently targeted in SRP-deficient cells may explain why previous genetic and biochemical analyses in yeast and bacteria did not reveal components of the SRP-dependent protein targeting pathway.

Targeted tumor cell internalization and imaging of multifunctional quantum dot-conjugated immunoliposomes in vitro and in vivo.

Targeted drug delivery systems that combine imaging and therapeutic modalities in a single macromolecular construct may offer advantages in the development and application of nanomedicines. To incorporate the unique optical properties of luminescent quantum dots (QDs) into immunoliposomes for cancer diagnosis and treatment, we describe the synthesis, biophysical characterization, tumor cell-selective internalization, and anticancer drug delivery of QD-conjugated immunoliposome-based nanoparticles (QD-ILs). Pharmacokinetic properties and in vivo imaging capability of QD-ILs were also investigated. Freeze-fracture electron microscopy was used to visualize naked QDs, liposome controls, nontargeted QD-conjugated liposomes (QD-Ls), and QD-ILs. QD-ILs prepared by insertion of anti-HER2 scFv exhibited efficient receptor-mediated endocytosis in HER2-overexpressing SK-BR-3 and MCF-7/HER2 cells but not in control MCF-7 cells as analyzed by flow cytometry and confocal microscopy. In contrast, nontargeted QD-Ls showed minimal binding and uptake in these cells. Doxorubicin-loaded QD-ILs showed efficient anticancer activity, while no cytotoxicity was observed for QD-ILs without chemotherapeutic payload. In athymic mice, QD-ILs significantly prolonged circulation of QDs, exhibiting a plasma terminal half-life ( t 1/2) of approximately 2.9 h as compared to free QDs with t 1/2 < 10 min. In MCF-7/HER2 xenograft models, localization of QD-ILs at tumor sites was confirmed by in vivo fluorescence imaging.

Pharmacological brake-release of mRNA translation enhances cognitive memory.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI: http://dx.doi.org/10.7554/eLife.00498.001

Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015).

The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.

Picomolar sensitivity MRI and photoacoustic imaging of cobalt nanoparticles

Multimodality imaging based on complementary detection principles has broad clinical applications and promises to improve the accuracy of medical diagnosis. This means that a tracer particle advantageously incorporates multiple functionalities into a single delivery vehicle. In the present work, we explore a unique combination of MRI and photoacoustic tomography (PAT) to detect picomolar concentrations of nanoparticles. The nanoconstruct consists of ferromagnetic (Co) particles coated with gold (Au) for biocompatibility and a unique shape that enables optical absorption over a broad range of frequencies. The end result is a dual-modality probe useful for the detection of trace amounts of nanoparticles in biological tissues, in which MRI provides volume detection, whereas PAT performs edge detection.

Resiliency and Vulnerability in the HER2-HER3 Tumorigenic Driver

The ability of certain breast cancers to resist a tyrosine kinase inhibitor drug may be overcome with high intermittent doses. How to Outsmart Breast Cancer Patients with breast cancer enrolled in recent clinical trials of a drug called lapatinib had reason to be optimistic. The growth and metastasis of many breast cancers depend critically on the target of this drug, the Erb receptor human epidermal growth factor 2 (HER2), and it made sense that its inhibition would hobble the cancer’s ability to survive. But some of these patients were ultimately disappointed as only a fraction of cancers responded to the drug, and those responses tended to be partial and transient. New work by Amin et al. in human breast cancer cells tests alternative treatment strategies and suggests that one of these might outwit these cancers. In certain breast tumors, the protein kinase activity of HER2, which is blocked by lapatinib, signals to downstream targets that cause cancer. One of these targets is another member of the same family, HER3, which can bind ligand but does not have catalytic activity of its own, and which in turn activates phosphoinositide 3-kinase (PI3K)–Akt signaling. In previous work, Amin and colleagues showed in human breast cancer cells that drug-induced altered regulation of HER3 through feedback from Akt is responsible for allowing cell to escape the lethal effects of lapatinib. Here, they probe this effect further and try to find a way to bypass the cells’ compensatory mechanism. The first approach was to try to inhibit PI3K at the same time as HER2 tyrosine kinase, but this proved ineffective as these cells were also able to up-regulate the growth signaling pathways and bypass inhibition by this combined treatment. Next, they used much higher doses of lapatinib, which were in fact able to completely and durably extinguish HER2 activity, but which have the disadvantage of being very toxic in vivo. They found a way around this problem by giving these high doses to mice with HER2-dependent tumors on an intermittent schedule, periodically driving blood concentrations high enough to generate a wave of apoptosis in the tumor and effectively preventing growth. The success of these authors in this skirmish with breast cancer marks a reason for renewed optimism in patients with HER2-dependent breast cancer. These second-generation approaches will need to be tested in the clinic, but the HER2-HER3 tumorigenic driver still seems to be an opponent keeping in our sites. About 25% of breast cancers harbor the amplified oncogene human epidermal growth factor receptor 2 (HER2) and are dependent on HER2 kinase function, identifying HER2 as a vulnerable target for therapy. However, HER2-HER3 signaling is buffered so that it is protected against a nearly two-log inhibition of HER2 catalytic activity; this buffering is driven by the negative regulation of HER3 by Akt. We have now further characterized HER2-HER3 signaling activity and have shown that the compensatory buffering prevents apoptotic tumor cell death from occurring as a result of the combined loss of mitogen-activated protein kinase (MAPK) and Akt signaling. To overcome the cancer cells’ compensatory mechanisms, we coadministered a phosphoinositide 3-kinase–mammalian target of rapamycin inhibitor and a HER2 tyrosine kinase inhibitor (TKI). This treatment strategy proved equivocal because it induced both TKI-sensitizing and TKI-desensitizing effects and robust cross-compensation of MAPK and Akt signaling pathways. Noting that HER2-HER3 activity was completely inhibited by higher, fully inactivating doses of TKI, we then attempted to overcome the cells’ compensatory buffering with this higher dose. This treatment crippled all downstream signaling and induced tumor apoptosis. Although such high doses of TKI are toxic in vivo when given continuously, we found that intermittent doses of TKI administered to mice produced sequential cycles of tumor apoptosis and ultimately complete tumor regression in mouse models, with little toxicity. This strategy for inactivation of HER2-HER3 tumorigenic activity is proposed for clinical testing.

Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional expression.

The signal recognition particle (SRP) is an evolutionarily conserved ribonucleoprotein (RNP) complex that functions in protein targeting to the endoplasmic reticulum (ER) membrane. Only two protein subunits of the SRP, Srp54p and Sec65p, and the RNA subunit, scR1, were previously known in the yeast Saccharomyces cerevisiae. Purification of yeast SRP by immunoaffinity chromatography revealed five additional proteins. Amino acid sequencing and cloning of the genes encoding four of these proteins demonstrated that the yeast SRP contains homologs (termed Srp14p, Srp68p and Srp72p) of the SRP14, SRP68 and SRP72 subunits found in mammalian SRP. The yeast SRP also contains a 21 kDa protein (termed Srp21p) that is not homologous to any protein in mammalian SRP. An additional 7 kDa protein may correspond to the mammalian SRP9. Disruption of any one of the four genes encoding the newly identified SRP proteins results in slow cell growth and inefficient protein translocation across the ER membrane. These phenotypes are indistinguishable from those resulting from the disruption of genes encoding SRP components identified previously. These data indicate that a lack of any of the analyzed SRP components results in loss of SRP function. ScR1 RNA and SRP proteins are at reduced levels in cells lacking any one of the newly identified proteins. In contrast, SRP components are present at near wild type levels and SRP subparticles are present in cells lacking either Srp54p or Sec65p. Thus Srp14p, Srp21p, Srp68p and Srp72p, but not Sec65p or Srp54p, are required for stable expression of the yeast SRP.

Building 'validated' mouse models of human cancer.

As a model system for the understanding of human cancer, the mouse has proved immensely valuable. Indeed, studies of mouse models have helped to define the nature of cancer as a genetic disease and demonstrated the causal role of genetic events found in tumors. As the scientific and medical communitys understanding of human cancer becomes more sophisticated, however, limitations and potential weaknesses of existing models are revealed. How valid are these murine models for the understanding and treatment of human cancer? The answer, it appears, depends on the nature of the research requirement. Certain models are better suited for particular applications. Using novel molecular tools and genetic strategies, improved models have recently been described that accurately mimic many aspects of human cancer.