May 1, 2014 (Vol. 34, No. 9)

In a complex system of communication, cells interact with their environment and other cells via signaling pathways.

Signaling pathways involve a variety of biochemical messengers that bind to protein receptors, initiating cellular responses. When these pathways turn awry, whether by gene mutation or dysregulation, the all-too-frequent result is tumorigenesis.

Identifying the mechanisms by which tumors circumvent certain signaling pathways will help develop an understanding of tumorigenesis and how tumors develop resistance to targeted therapies. Advancements in cell signaling will be discussed at “Protein Phosphorylation, Cellular Plasticity, and Signaling Rewiring,” FASEB Science Research Conference scheduled for July 2014.

The MET gene, encoding the hepatocyte growth factor (HGF) receptor, is widely expressed in many solid tumor types and in some hematologic malignancies. Although MET gene amplification and mutation, both potentially oncogenic events, occur in solid tumors at a relatively low frequencies, MET overexpression is common and also related to pathway activation. HGF/MET autocrine signaling, also oncogenic, occurs at a low but measurable frequency.

Because the pathway normally contributes to embryologic development and morphogenesis, it can also contribute later in cancer progression to tumor invasiveness and metastasis of various solid tumor types. A rare inherited form of human kidney cancer, known as hereditary papillary renal carcinoma (HPRC), suggests that the pathway can initiate a deadly malignancy.

HPRC patients respond to MET-targeted therapy profoundly and at a high rate. In that population, it is a singular oncogenic driver, and therefore single-agent therapy with a MET inhibitor is likely to be efficacious. In other cancers, it may play a critical but not singular role. The HGF/MET pathway shares downstream effectors with other well-known oncogenic receptor tyrosine kinases, and therefore can exacerbate oncogenesis initiated by them.

According to Donald P. Bottaro, Ph.D., senior scientist, Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, the pathway can be targeted with conventional strategies such as neutralizing antibodies that block ligand-receptor interactions and ATP-competitive small molecule kinase inhibitors.

In addition, the pathway can be targeted using small molecule SH2 domain-binding antagonists, as well as using an engineered ligand variant that disrupts critical heparin sulfate-mediated MET-HGF-co-receptor interactions. Research in other laboratories has shown that selective disruption of non-SH2 domain-mediated receptor effector interactions or primary effector function can also inhibit the pathway.

A spectrum of inhibitory strategies has proven valuable in oncology in the past, and, in most cases, targeting of the HGF/MET pathway is likely to be combined with conventional therapeutic strategies and/or other molecularly targeted agents. Identifying cancer patients most likely to benefit from treatment that includes HGF/MET pathway inhibitors depends critically on pathway-related diagnostic biomarker development currently in progress.

Researchers at the Center for Cancer Research, National Cancer Institute, are exploring intervention strategies that impact on the HGF/MET pathway. This pathway shares downstream effectors with other well-known oncogenetic receptor tyrosine kinases.

Lung Cancer Pathways

Receptor tyrosine kinases (RTKs) are known drivers of lung cancer cell survival and proliferation, and they can be activated through both genomic as well as tumor microenvironment mechanisms.

Targeting genomic alterations in RTKs, such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), with small molecule tyrosine kinase inhibitors (TKIs) can have major effects on patient survival, and they are a key target for therapeutics. For example, erlotinib hydrochloride targets EGFR, and crizotinib targets ALK.

In lung cancer, tumors can evade death by tyrosine kinase inhibition through various genomic and stromal mechanisms. For example, second-site mutations in EGFR can block binding of erlotinib, and tumors can regrow despite the small molecule’s presence.

Work from multiple labs has shown that growth factors produced by stromal cells in the microenvironment can drive resistance, supporting the idea of targeting multiple RTKs. Molecular tools that can identify aberrant RTKs on tumor cells can guide clinical decision making for oncologists and their patients.

“We hypothesized that measurement of signaling-associated complexes of RTKs may be able to mark RTK-driven tumors. RTK activation results in binding of proteins to facilitate signal transduction. This includes adaptor proteins, such as Grb2 or Shc1, that are used to couple growth-factor signaling to the MAPK pathway and drive cancer cell proliferation,” discusses Eric B. Haura, M.D., senior member, Moffitt Cancer Center.

“Using mass spectrometry along with affinity purification, we have defined complexes that reflect an activated RTK.”

Mutant forms of EGFR have been shown to have different components of the protein complex compared to wild-type forms. To capitalize on this knowledge, proximity ligation assays (PLAs) were employed to characterize EGFR:GRB2 protein complexes in situ in FFPE tumor tissues, allowing in situ analysis of signaling complexes across many unique tumors.

Tumors that have activated EGFR signaling-associated complexes, measured by EGFR:GRB2 PLA, have better response to EGFR-targeting agents.

“Measuring signaling complexes directly in human tumor tissues helps us understand the plasticity of tumor cells, how they rewire in response to therapeutics, as well as tumor heterogeneity at the signaling level,” concludes Dr. Haura.

Researchers at the Moffitt Cancer Center have defined complexes that reflect an activated receptor tyrosine kinase. In this image, EGFR:GRB2 signaling-associated protein complexes (red) are shown in a human lung adenocarcinoma (green) tissue specimen detected by proximity ligation assay.

Breast Cancer Pathways

ErbB-3, an EGFR family member that is thought to be significant in breast cancer progression, is the major receptor that recruits the phosphatidylinositol 3′ kinase (PI3K) signaling cascade to ErbB-2, a major oncogene in induction of mammary cancer. PI3K is a lipid kinase involved in the generation of a lipid intermediary important in activating a number of downstream serine protein kinases, the AKT family.

To investigate the role of ErbB-3, a transgenic mouse that expresses activated ErbB-2 in the mammary epithelium—these mice develop metastatic mammary tumors—was crossbred to a mouse genetically engineered to lack the binding sites for the PI3K binding site on ErbB-3.

The results were equivocal. While there was an effect on normal mammary gland development by knocking out the PI3K binding sites on ErbB-3, there was a relatively modest effect on ErbB-2 tumor induction. This may be due to the ability of the other EGF receptor family members to recruit this PI3K pathway.

Although ErbB-3 is being therapeutically targeted, these results suggest this strategy may ultimately fail. In many instances, inhibitors directed against different signaling components fail because of compensatory signaling pathways.

Additionally, the role of the first oncogene discovered in cancer, c-Src, has always been unclear. A transgenic mouse model, the polyoma middle-T mouse, was used to generate a traditional knockout of c-Src, enabling specific ablation of the c-Src function in the mammary epithelium using a second transgene expressing a mammary-specific Cre.

This produced a dramatic effect on tumor induction that seems to be involved in regulating another gene product, p27, a key cell cycle regulator. Normally, c-Src inactivates this break in cell cycle and allows cells to progress; when c-Src is lost, this break gets automatically turned back on and the cells cannot proliferate.

“The critical advantage of transgenic mouse model systems is that they have a completely normal immune microenvironment,” explains William Muller, Ph.D., professor of biochemistry, McGill University. “These systems are primed to develop tumors and undergo the natural history of tumor development, not just a tumor end-stage phenomenon. You can look at drug effects on different stages.”


Almost all aspects of biology are regulated by reversible protein phosphorylation and ubiquitination.

Protein ubiquitination is analogous to protein phosphorylation except that ubiquitin molecules are attached covalently to Lys residues, as opposed to phosphate groups becoming covalently attached to one or more Ser, Thr, or Tyr residues. Like phosphorylation, ubiquitination can alter protein properties and functions, and is likely to be a more versatile control mechanism, as ubiquitin molecules cannot only be linked to one or more amino acid residues on the same protein, but can also form ubiquitin chains.

Although the identities of most of the proteins that are likely to be players in regulating ubiquitination pathways are known, there is little information on which components in these pathways would represent the best targets for treatment of disease. The Cooper Lab at the Fred Hutchinson Cancer Research Center studies the particular case where Cullin-5-based ubiquitin ligases oppose Src kinase-activated signaling, using two systems.

The first one, an in vivo system using genetic manipulation, studies the migrations of neurons that take place during brain development. The second system uses cultured epithelial cells to facilitate detailed cell biology. The finding in both cases is that Cullin-5-based ligases inhibit Src functions, but the mechanisms are quite different.

“The challenges we face in our systems are the small biochemical effects, yet large biological effects,” observes Jonathan A. Cooper, Ph.D., principal investigator. “We think that the specific ubiquitination of only phosphorylated molecules, coupled with the typically low phosphorylation stoichiometry of the substrates, means that net turnover rates are only slightly altered.”

“This creates enormous technical challenges in identifying substrates by classic approaches. However, the biology can be profound. Does this mean that the biology is not caused by the ubiquitination and degradation? Perhaps, but maybe there are more interesting explanations.”

“There is interest in proteasome inhibitors, and a general Cullin inhibitor that is useful in the lab might be useful in the clinic. However, developing specific inhibitors for individual ubiquitin ligases is very tricky, as has been the case for inhibiting individual protein kinases,” states Dr. Cooper. “Finding the right target needs basic research into mechanisms and pathways.”

The Cooper Lab at the Fred Hutchinson Cancer Research Center has shown that Cullin-5 suppresses epithelial cell migration. Top: Scratch wound-healing assays. Bottom: Immunofluorescent images of migrating cells at the edge of a wound. Cullin-5-deficient cells have an elongated leading lamellipodium and tiny focal adhesions.

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