Archive for the ‘Kinase’ Category

Moffitt Cancer Center researchers identify drivers of sarcoma growth and survival

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Posted 06 May 2012 — by James Street
Category Kinase, Osteosarcoma, Proteomics, Sarcoma, Tyrosine Kinase

Posted On: May 1, 2012 – 9:00pm

To better understand the signaling pathways active in sarcomas, researchers at Moffitt Cancer Center used state-of-the-art mass spectrometry-based proteomics to characterize a family of protein enzymes that act as “on” or “off” switches important in the biology of cancer. The tyrosine kinases they identified, the researchers said, could act as “drivers” for the growth and survival of sarcomas.

Sarcomas are relatively rare forms of cancer. In contrast to carcinomas, which arise from epithelial cells (in breast, colon and lung cancers, for example), sarcomas are tumors derived from bone, fat, muscle or vascular tissues.

“Sarcomas are rare, diverse malignancies that arise from connective tissues,” said study lead author Eric B. Haura, M.D., program leader for Experimental Therapeutics. “We hypothesized that we could identify important proteins that drive the growth and survival of these poorly understood sarcomas using an approach to characterize signaling proteins using mass spectrometry.”

According to Haura, whose lab focuses on signaling pathways in cancer and understanding the role of kinases, protein phosphorylation plays a significant role in a wide range of cellular processes and is commonly disrupted in diseases such as cancer. The study approach is novel by engaging proteomics, an emerging and increasingly powerful approach to study proteins in disease in a more global and unbiased manner.

In this study, the Moffitt researchers identified 1,936 unique tyrosine phosphorylated peptides corresponding to 844 unique phospho-tyrosine proteins and found 39 tyrosine kinases in sarcoma cells. Of the 99 tyrosine kinases present in the human genome, the research team identified peptides corresponding to nearly 40 percent of the tyrosine kinome.

“Tyrosine kinases play an important role in controlling the hallmarks of cancer, and they have a proven track record as druggable targets for cancer treatment. Our goal was to produce a ‘landscape’ of tyrosine phosphorylated proteins and tyrosine kinases prioritized for subsequent functional validation,” Haura said. “In our study, we identified numerous tyrosine kinases that can be important for cellular signaling in human sarcomas that could drive the natural progression of sarcoma and, therefore, could be targeted by small molecule inhibitors aimed at altering sarcoma progression.”

Questions remain, however, about which, if any, of the 40 tyrosine kinases the researchers identified in sarcoma tumor cell lines act to regulate sarcoma tumor cell growth and tumor survival.

“The answers to this question can help prioritize which potential targets to examine further, including advancement into trials of patients with advanced sarcoma,” explained Haura. “As a first step, we screened sarcoma cell lines against a number of inhibitors selected, all based on the tyrosine kinases we identified, and identified some active drugs.”

While the researchers found kinases in sarcoma cells that deserved further study, they also concluded that the sarcoma cells tested expressed multiple tyrosine kinases. That great number may limit the effectiveness of targeted agents.

“We think this approach could hold promise in profiling tumors directly from patients and can complement existing genetic data on sarcomas. Our results show this is feasible in tumor tissues, and we hope to advance this further by directly studying additional tumors from sarcoma patients.”

 

Studies Suggest Single Protein May Control Inflammation-Related Tumor Growth

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Posted 14 Jun 2011 — by James Street
Category Kinase, Kinome, Molecular, Proteomics

    Scientists say that they have discovered that a single protein may control tumor inflammation and progression. They claim that tumor- derived chemoattractants that stimulate myeloid cell receptor tyrosine kinases (RTKs), toll-like/IL-1 receptors (TLR/IL1Rs), and GPCRs unexpectedly activate a single PI3-kinase isoform known as p110γ and a single integrin called α4β1 to promote myeloid cell recruitment to tumors and tumor progression.

    The team, based at the University of California, San Diego (UCSD) and University of Torino in Italy, reports their findings in Cancer Cell. The paper is titled “Receptor Tyrosine Kinases and TLR/IL1Rs Unexpectedly Activate Myeloid Cell PI3Kg, A Single Convergent Point Promoting Tumor Inflammation and Progression.” Their work overturns the widely held tenet that p110γ is only activated by GPCRs, says USCD’s Judith A. Varner, Ph.D., and colleagues.

    The team first confirmed that CD11b+ myeloid cells are recruited to and extensively populate a range of human and murine tumors, including murine and human breast, pancreatic, and lung carcinomas but not corresponding normal tissues. These cells persistently invaded growing tumors over time until as much as 25% of a tumor’s mass comprised myeloid cells.

    Furthermore, tumor inflammation was directly proportional to angiogenesis throughout the growth of the tumor. Additional studies showed that inflammatory factors in the tumor microenvironment were derived from both tumor and inflammatory cells.

    As most tumors produce multiple chemoattractants, the researchers then looked to see whether a common mechanism was involved in regulating myeloid cell recruitment to tumors. They tested the ability of chemoattractants to promote myeloid cell adhesion to endothelial cells (ECs) in vivo and in vitro.

    They took this approach because in order to exit the blood stream in response to signals released from diseased tissues, immune cells transiently adhere to and transmigrate through vascular endothelium, a process that depends on adhesion to EC receptors. A diverse range of tumor-derived chemoattractants was able to tigger EC adhesion by myeloid cells.

    Previous studies have shown that the myeloid cell receptors α4β1 and αMβ2 are involved in recognizing the EC surface adhesion proteins VCAM-1 and ICAM-1 and that blocking α4β1 but not αMβ2 suppresses murine and human myeloid cell adhesion to ECs. Dr. Varner’s team confirmed that murine myeloid cells in which integrin α4 expression was ablated by siRNA also failed to adhere to EC and the EC surface adhesion protein VCAM-1.

    “As chemoattractants had no effect on EC expression of α4 ligands during these assays, these results indicate that diverse chemoattractants stimulate myeloid cell adhesion by selectively increasing integrin α4β1 but not αMβ2,” the authors note.

    Indeed, further investigation indicated that chemokines, cytokines, and growth factors that activate RTKs, TLR/IL1Rs, and GPCRs all activate integrin α4β1, thereby promoting murine and human myeloid cell adhesion to ECs. Importantly, knocking out integrin α4 in tumors implanted in mice provide evidence that integrin α4 activation is required for trafficking and infiltration of myeloid cells into tumors in vivo.

    The observation that the chemoattractants stimulating structurally diverse GPCRs, RTKs, TLR/IL1Rs, and type I cytokine receptors all activate myeloid cell integrin α4β1 indicates that a common downstream signaling pathway might link these receptors, the authors write. When they evaluated inhibitors of various signaling pathways using myeloid cell adhesion assays, they found that selective inhibitors of PI3Kγ (p110γ) and Ras GTPases blocked myeloid cell adhesion to endothelium, while inhibitors of other PI3K isoforms and signaling proteins had no effect on adhesion.

    The class IB PI3K p110γ has been well-characterized as a GPCR-activated lipid kinase that promotes chemokine-stimulated chemotaxis and polarization of neutrophils, lymphocytes, and thymocytes in vitro and in vivo. p110γ-knockout mice and animals that express an inactivated form of p110γ have been shown to exhibit defects in granulocyte responses to chemokines.

    Previous studies have in addition shown that p110γ is essential for GPCR- but not RTK-mediated activation of PI3K activity, which makes the new observation that p110γ inhibitors blocked adhesion in response to GPCR as well as RTK ligands particularly unexpected, the team stresses.

    Using p110γ-specific knockout or inhibition methods, the researchers went on to demonstrate that growth factors, interleukins, and chemokines, which are ligands for RTKs, TLR/IL-1Rs, and GPCRs, promoted adhesion of wild-type, but not of P110γ-knockout myeloid cells or those with inactive P110γ to ECs or VCAM-1. Given that the knockdown cells still expressed normal levels of α4 integrin, these results indicated that p110γ is necessary for integrin adhesive activity. siRNA knockdown of p110γ but not other PI3K catalytic subunits also suppressed adhesion to ECs or VCAM-1, regardless of the stimulus, the authors add.

    “Although prior studies have indicated that only GPCR ligands activate p110γ, our results indicate that ligands for RTKs and TLR/IL1Rs promote p110γ activity and p110γ-dependent integrin α4β1 activation,” they claim. “Importantly, p110γ is also sufficient for integrin α4β1 activation as cells from p110γCAAX mice, which express membrane-targeted, constitutively activated p110γ adhered strongly to ECs even in the absence of stimulation.”

    In a further set of studies the team went on to confirm the pivotal role of p110γ but not other PI3K isoforms in promoting α4β1-mediated myeloid cell trafficking to tumors in vivo. They surprisingly found that p110γ was the major PI3K catalytic isoform expressed in primary myeloid cells, whereas lymphocytes express large amounts of p110δ, while LLC cells express large amounts of p110α and β and little γ or δ. Additional in vitro studies demonstrated that growth factors and cytokines activate p110γ directly, rather than indirectly through GPCRs.

    The combined collection of findings made particular sense when viewed in a clinical context. The researchers tested  the efficacy of a selective p110γ inhibitor or a pan-PI3K inhibitor, in tumor-bearing mice. While both inhibitors suppressed lung cancer inflammation angiogenesis and tumor growth  in vivo, activity of the P110γ inhibitor was related to the inhibition of tumor inflammation and angiogenesis, without directly affecting the tumor cells themselves.

    While a number of clinical trials are already under way to test pan-PI3K inhibitors in cancer patients, “our studies indicate that p110γ is an excellent target for a relatively nontoxic cancer therapeutic, as this isoform is primarily expressed by myeloid cells and is a convergence point of diverse chemoattractant signaling pathways that are required for tumor inflammation and tumor progression,” the authors conclude.

PX-866 Targets the Phosphatidylinositol-3-kinase (PI-3K) Pathway

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Posted 13 Jun 2011 — by James Street
Category Drugs, Kinase, Kinome, Proteomics, PTEN

PX-866 is a small molecule drug that irreversibly inhibits phosphatidylinositol-3-kinase (PI-3K), a family of intracellular signaling molecules that play essential roles in multiple cellular functions. In normal cells the PI-3K pathway is tightly controlled, whereas the inappropriate activation of PI-3K is important in the pathogenesis of multiple human cancers, making it a rational and attractive target for drug development.

There are 3 classes of PI-3K (I, II and III) categorized based on structure and substrate specificity. The class I PI-3Ks are most tightly associated with human disease and are the targets of PX-866. Four class I PI-3K isoforms have been identified: alpha (α), beta (β), delta (δ) and gamma (γ). The α and β PI-3Ks are broadly expressed in human tissues and deregulation of these two PI-3K family members occurs in many solid tumors. The δ and γ PI-3Ks are primarily expressed in cells that comprise the human immune system and the δ PI-3K is important for lymphomas and leukemia tumor cell growth.

In normal cells the class I PI-3Ks are activated by the stimulation of signaling receptors found on the cell surface that respond to soluble growth factors or cell-cell contact. Once activated, class I PI-3K chemically modifies a membrane-associated lipid molecule called phosphatidylinositol 4,5-bisphosphate (PIP2) to form the product phosphatidylinositol 3, 4, 5-trisphosphate (PIP3). PIP3 serves as a “second messenger” that activates downstream targets, including AKT, the principal mediator of PI-3K signal transduction. The activation of AKT triggers the subsequent activation of additional downstream signaling molecules including the mammalian target of rapamycin (mTORC1), as well as other proteins that control cell growth, metabolism, survival, and proliferation.

In non-diseased cells the PI-3K pathway is tightly controlled by a molecule called PTEN, which counteracts the activity of class I PI-3K by converting PIP3 back to PIP2. Many cancers contain mutations that result in the deregulated activation of the PI-3K pathway. These mutations may function by inhibiting the activity of PTEN, or by overcoming PTEN activity through enhancement of PI-3K signaling. This enhancement can occur by mutations within the PI-3K gene that result in increased enzymatic activity, or by changes to the growth factor receptors that are upstream of PI-3K. These receptors include EGFR, cMet, IGF1R and Her2, which have been shown to be important for multiple cancers. The deregulated activation of PI-3K drives multiple biological activities that are critical for cancer cells, including processes that enable tumors to metastasize and invade normal tissues, continue to grow under conditions of low nutrients or low oxygen, and to resist chemotherapy and radiation therapy (Engelman 2009; Liu, Cheng et al. 2009; Sarker, Reid et al. 2009; Ghayad and Cohen 2010).

Preclinical studies have shown that PX-866 is efficacious in numerous mouse xenograft models of human cancers as a single agent and in combination with chemotherapy, radiation and targeted cancer drugs, such as EGFR inhibitors. Oncothyreon is currently evaluating PX-866 in Phase 1 / 2 and Phase 2 clinical studies in solid tumor diseases.

Promising new target for stifling the growth and spread of cancer

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Posted 13 Jun 2011 — by James Street
Category Inflamation, Kinase, Kinome, metastases, Proteomics

UCSD researchers find inhibiting single protein blocks the inflammation that fuels tumors

Cancer and chronic inflammation are partners in peril, with the latter increasing the likelihood that malignant tumors will develop, grow and spread. Researchers at the University of California, San Diego School of Medicine say they’ve identified a tumor inflammation trigger that is common to most, if not all, cancers. And using existing inhibitory drugs, the scientists were able to dramatically decrease primary tumor growth in animal studies and, more importantly, halt tumor progression and metastasis.

The findings appear in the June 14 issue of the journal Cancer Cell, authored by Judith A. Varner, PhD, professor of medicine at the UC San Diego Moores Cancer Center, and colleagues in the UCSD School of Medicine and at the University of Torino, Italy.

When cancer cells appear in the body, they often provoke an immune system response. Under some circumstances, this is a good thing. But Varner and colleagues were able to show that when responding myeloid or white blood cells called macrophages are drawn to invasive cancer cells, the result can be considerable trouble for patients. Rather than suppressing the cancer, the myeloid cells are tricked by the tumor into aiding and abetting its growth and spread. Scientists have long recognized that myeloid cells can invade and promote tumor growth. But until now it was not fully appreciated how this hijacking occurs and whether there are ways to disrupt this process by suppressing the trigger that leads to myeloid cell recruitment into tumors.

Probing more deeply into the tumor inflammation process, the UCSD research team identified a range of tumor-produced molecules that attract these dangerous myeloid cells. They also pinpointed the specific trigger on myeloid cells enabling them to invade the tumor environment and accelerate tumor growth and metastasis. It is an enzyme called PI-3 kinase gamma on myeloid cells that turns on an adhesion receptor allowing the cells to enter tumors.

When researchers blocked the activity of PI-3-kinase-gamma, either genetically or through the use of a drug designed for this purpose, myeloid cells were blocked access into tumors, resulting in reduced tumor growth and a dramatic decrease in metastasis. Without the recruitment of myeloid cells, Varner said, the capability of a cancer tumor to grow is largely stifled.

“Most strategies targeting the role of myeloid cells in cancer have focused on reducing their expression of inflammatory molecules,” Varner explained. “We’ve found a single convergent point – the PI-3 kinase-gamma enzyme – that, when blocked, appears to result in significant suppression of tumor inflammation and growth regardless of the initiating event. It could be a very important therapeutic target for future cancer treatments and could impact most, if not all, types of solid cancer.”

Michael Karin, PhD, distinguished professor of pharmacology in UCSD’s Laboratory of Gene Regulation and Signal Transduction and a pioneer in inflammation research, agreed: “I think that the inhibition of PI-3K activity represents a very interesting and promising approach for inhibition of tumor-associated inflammation. It seems to fully normalize the tumor microenvironment and provide a new addition to our armamentum of anti-cancer drugs.”

Varner said a number of biotechnology companies are pursuing potential drugs using PI-3-kinase inhibitors to treat diseases from cancer to heart disease to arthritis. The PI-3-kinase-gamma protein may be a particularly promising therapeutic target, because it is not widely expressed in the body, and its inhibition would likely produce fewer side effects than many therapeutics.

###

 

Co-authors of the research are Michael C. Schmid, Christie J. Avraamides, Philippe Foubert, Joan R.E. Manglicmot, Xiaodan Song and Wolfgang Wrasidlo of the UCSD Moores Cancer Center; Holly C. Dippold and Mark H. Ginsberg, UCSD Department of Medicine; Irene Franco and Emilio Hirsch, Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, School of Medicine, University of Torino, Italy; Lesley G. Ellies, UCSD Department of Pathology; Sara L. Blair, UCSD Department of Surgery; and Lissette M. Acevedo and David A. Cheresh, UCSD Moores Cancer Center and UCSD Department of Pathology.

Funding for this research came, in part, from grants from the National Institutes of Health and the California Tobacco Related Disease Research Program.

Contact: Scott LaFee
slafee@ucsd.edu
619-543-6163 begin_of_the_skype_highlighting 619-543-6163 end_of_the_skype_highlighting
University of California – San Diego

New Kinome Database Promises Wealth of Unexpected Cancer Drug Targets

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Posted 07 Jun 2011 — by James Street
Category Kinase, Kinome
By Lois Wingerson | January 5, 2011

 

A word for oncologists to know this year (if they don’t already) is “kinome.” Analagous to “genome,” it’s the complete set of kinases in an organism. The human kinases, of course, are the class of enzymes whose inhibitors have proven so promising and so frustrating in recent clinical cancer trials.

Over the past year or so, study of the kinome has revealed molecules that some tumors are relying on to survive, many of which until now we had no idea were involved in cancer or which we hardly knew at all. Numerous reports have suggested novel kinases that might be useful drug targets in particular malignancies.

In September, for instance, a team from Leiden in the Netherlands reported in Molecular Cancer that they had used kinome analysis to identify two kinases (Src and nuclear factor kappaB) involved in myxoid liposarcoma. Earlier last year, two different teams in the United States found new kinases involved in the development of ovarian cancer and in its response to cis-platin treatment.

From tidbits to a feast

Other teams have been serving up tidbits about new kinases, piecemeal. Now a team from Finland has offered drug developers a feast: A “functional taxonomy” of the kinome, revealing which of 459 kinase proteins show either significant gains or losses of activity in human tumors compared to normal tissues. The data are based on analysis of 5,681 human tissue samples.

As an appetizer, the report by Sami Kilpinen et al in PLoS One last month provides the identities of several hitherto obscure kinases that appear to be involved in prostate and gynecological cancers as well as some lymphomas. It identifies altogether 22 kinases not previously associated with prostate cancer. Far more is certain to come, because the database is published openly on the Internet.

More than 10,000 patent applications for kinase inhibitors have been filed in the last decade in the United States, because many of the 518 human kinases and other molecules in their pathways are among the most frequently mutated in cancer. Although kinase inhibitors have revolutionized the treatment of a few cancers such as chronic myeloid leukemia (CML) and gastrointestinal stromal tumor (GIST), “clinical progress has been uneven,” Zachary Knight of Rockefeller University and coauthors observed in a review about “kinomics” and cancer in Nature Reviews Cancer last February.

Kinase inhibitors have achieved significant survival gains for only a few cancers, because relatively few malignancies are “addicted” to only one kinase mutation for their survival. Many develop resistance to a particular kinase inhibitor by mutating, reverting to a different kinase signaling pathway, or making molecular adjustments downstream of the inhibitor. Rationally designed combinations of kinase inhibitors have failed in malignancy after malignancy, at least in part because of insufficient understanding of the kinome.

Expression patterns match side effects

The mass of information from Kilpinen et al also promises to offer insights into the side effects that have hindered widespread use of some kinase inhibitors, because it shows kinase expression patterns for a wide range of normal tissues. The skin rash associated with epidermal growth factor receptor (EGFR) is echoed in the discovery of elevated transcriptional activity in hair follicles in the Finnish study. ERBB2/HER-2 was shown to be active in healthy tissues including heart, lung, and colorectal tissues, corresponding to the most common side effects of anti-ERBB2 drugs: cardiovascular and respiratory problems and diarrhea.

The dataset also contributes important information by revealing co-expression of different kinases in similar tumors, offering insights for combination therapies. As Knight et al observed in their review of the field, “There are clearly clusters of kinases that tend to be inhibited by similar drugs, but … there are also many target combinations that should be accessible but remain undiscovered.”

They speculate that some pharmaceutical companies already try to optimize drug profiles against complex patterns of kinases, rather than individually, in order to maximize their specificity. The public availability of the Finnish data can only speed this process.

WEE1 inhibition sensitizes Osteosarcoma to Radiotherapy

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Posted 29 Apr 2011 — by James Street
Category Kinase, Molecular, Radiation

The use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor.

After irradiation-induced DNA damage, OS cells sustain a prolonged G2 cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G2 arrest and could sensitize OS cells to irradiation induced cell death.

Methods: WEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples.

WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment.

The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.

Results: WEE1 expression is found in the majority of tested OS tissue samples.

Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation.

The inhibition of WEE1 effectively abrogates the irradiation-induced G2 arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.

Conclusion: We show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G2 checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS.

Author: Jantine PosthumaDeBoer Thomas Wurdinger Harm Graat Victor van Beusechem Marco Helder Barend van Royen Gert-Jan Kaspers
Credits/Source: BMC Cancer 2011, 11:156