Michael Reiss

ADAMTS1 and MMP1 proteolytically engage EGF-like ligands in an osteolytic signaling cascade for bone metastasis

Bone metastasis is mediated by complex interactions between tumor cells and resident stromal cells in the bone microenvironment. The functions of metalloproteinases in organ-specific metastasis remain poorly defined despite their well-appreciated role in matrix degradation and tumor invasion. Here, we show a mechanism whereby two distinct metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1) and matrix metalloproteinase-1 (MMP1), orchestrate a paracrine signaling cascade to modulate the bone microenvironment in favor of osteoclastogenesis and bone metastasis. Proteolytic release of membrane-bound epidermal growth factor (EGF)-like growth factors, including Amphiregulin (AREG), heparin-binding EGF (HB-EGF), and transforming growth factor alpha (TGFalpha) from tumor cells suppress the expression of osteoprotegerin (OPG) in osteoblasts and subsequently potentiate osteoclast differentiation. EGF receptor (EGFR) inhibitors block osteolytic bone metastasis by targeting EGFR signaling in bone stromal cells. Furthermore, elevated MMP1 and ADAMTS1 expression is associated with increased risk of bone metastasis in breast cancer patients. This study established MMP1 and ADAMTS1 in tumor cells, as well as EGFR signaling in osteoblasts, as promising therapeutic targets for inhibiting bone metastasis of breast cancer.

Phase I Study of GC1008 (Fresolimumab): A Human Anti-Transforming Growth Factor-Beta (TGFβ) Monoclonal Antibody in Patients with Advanced Malignant Melanoma or Renal Cell Carcinoma

Background In advanced cancers, transforming growth factor-beta (TGFβ) promotes tumor growth and metastases and suppresses host antitumor immunity. GC1008 is a human anti-TGFβ monoclonal antibody that neutralizes all isoforms of TGFβ. Here, the safety and activity of GC1008 was evaluated in patients with advanced malignant melanoma and renal cell carcinoma. Methods In this multi-center phase I trial, cohorts of patients with previously treated malignant melanoma or renal cell carcinoma received intravenous GC1008 at 0.1, 0.3, 1, 3, 10, or 15 mg/kg on days 0, 28, 42, and 56. Patients achieving at least stable disease were eligible to receive Extended Treatment consisting of 4 doses of GC1008 every 2 weeks for up to 2 additional courses. Pharmacokinetic and exploratory biomarker assessments were performed. Results Twenty-nine patients, 28 with malignant melanoma and 1 with renal cell carcinoma, were enrolled and treated, 22 in the dose-escalation part and 7 in a safety cohort expansion. No dose-limiting toxicity was observed, and the maximum dose, 15 mg/kg, was determined to be safe. The development of reversible cutaneous keratoacanthomas/squamous-cell carcinomas (4 patients) and hyperkeratosis was the major adverse event observed. One malignant melanoma patient achieved a partial response, and six had stable disease with a median progression-free survival of 24 weeks for these 7 patients (range, 16.4–44.4 weeks). Conclusions GC1008 had no dose-limiting toxicity up to 15 mg/kg. In patients with advanced malignant melanoma and renal cell carcinoma, multiple doses of GC1008 demonstrated acceptable safety and preliminary evidence of antitumor activity, warranting further studies of single agent and combination treatments. Trial Registration Clinicaltrials.gov NCT00356460

Targeting the Transforming Growth Factor-β pathway inhibits human basal-like breast cancer metastasis

BackgroundTransforming Growth Factor β (TGF-β) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-β antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-β pathway antagonists (1D11, a mouse monoclonal pan-TGF-β neutralizing antibody and LY2109761, a chemical inhibitor of TGF-β type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR).ResultsBoth 1D11 and LY2109761 effectively blocked TGF-β-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-β stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-β. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-β antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.ConclusionsIn aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.

Transforming growth factor-β signaling: emerging stem cell target in metastatic breast cancer?

In most human breast cancers, lowering of TGFβ receptor- or Smad gene expression combined with increased levels of TGFβs in the tumor microenvironment is sufficient to abrogate TGFβs tumor suppressive effects and to induce a mesenchymal, motile and invasive phenotype. In genetic mouse models, TGFβ signaling suppresses de novo mammary cancer formation but promotes metastasis of tumors that have broken through TGFβ tumor suppression. In mouse models of “triple-negative” or basal-like breast cancer, treatment with TGFβ neutralizing antibodies or receptor kinase inhibitors strongly inhibits development of lung- and bone metastases. These TGFβ antagonists do not significantly affect tumor cell proliferation or apoptosis. Rather, they de-repress anti-tumor immunity, inhibit angiogenesis and reverse the mesenchymal, motile, invasive phenotype characteristic of basal-like and HER2-positive breast cancer cells. Patterns of TGFβ target genes upregulation in human breast cancers suggest that TGFβ may drive tumor progression in estrogen-independent cancer, while it mediates a suppressive host cell response in estrogen-dependent luminal cancers. In addition, TGFβ appears to play a key role in maintaining the mammary epithelial (cancer) stem cell pool, in part by inducing a mesenchymal phenotype, while differentiated, estrogen receptor-positive, luminal cells are unresponsive to TGFβ because the TGFBR2 receptor gene is transcriptionally silent. These same cells respond to estrogen by downregulating TGFβ, while antiestrogens act by upregulating TGFβ. This model predicts that inhibiting TGFβ signaling should drive the differentiation of mammary stem cells into ductal cells. Consequently, TGFβ antagonists may convert basal-like or HER2-positive cancers to a more epithelioid, non-proliferating (and, perhaps, non-metastatic) phenotype. Conversely, these agents might antagonize the therapeutic effects of anti-estrogens in estrogen-dependent luminal cancers. These predictions need to be addressed prospectively in clinical trials and should inform the selection of patient populations most likely to benefit from this novel anti-metastatic therapeutic approach.

TGFBR1*6A and Cancer: A Meta-Analysis of 12 Case-Control Studies

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Loss of Smad signaling in human colorectal cancer is associated with advanced disease and poor prognosis.

PURPOSEBased largely on in vitro inve stigations and animal studies, investigators believe that disruptions of transforming growth factor-β (TGF-β) signaling contribute to the development and progression of human colorectal cancer. The purpose of this study was to directly assess the status of the TGF-β signaling pathway in colorectal cancer and determine the effects of its disruption on clinical behavior and outcome. MATERIALS AND METHODSSmad proteins are the principal intracellular components of the TGF-β signaling pathway. We conducted a high-throughput analysis of the expression patterns of Smad2, phosphorylated (activated) Smad2 (pSmad2), and Smad4 in more than 600 human colorectal cancer specimens assembled in tissue microarrays. RESULTSThe vast majority (93.8%; 95% Cl: 92%–96%) of colorectal cancers expressed phosphorylated Smad2, indicating the ability of the tumors to survive and proliferate within a microenvironment that contains bioactive TGF-β. Twelve of 633 (1.9%; 95% Cl: 1%–3%) cases failed to express Smad2, and 15 of 641 (2.3%; 95% Cl: 1%–4%) cases failed to express Smad4. Moreover, 29 of 615 (4.7%; 95% Cl: 3%–7%) of cases expressed Smad2 but not its activated form (pSmad2), suggesting the presence of a TGF-β receptor defect. Based on an analysis of 577 cases for which clinical outcome information was available, failure to express Smad2, pSmad2, or Smad4 was associated with advanced stage disease, the presence of lymph node metastases, and a significantly shorter overall survival (median survival: 35 vs 58 months). DISCUSSIONLoss of Smad activation and/or expression occurs in approximately 10% of colorectal cancers. This subset has a poor prognosis because of its association with advanced disease and the presence of lymph node metastases at diagnosis.

Novel inactivating mutations of transforming growth factor-β type I receptor gene in head-and-neck cancer metastases

Carcinoma cell lines are frequently refractory to transforming growth factor‐β (TGFβ)‐mediated cell cycle arrest. Whether and how TGFβ signaling is disrupted in the majority of human tumors, however, remains unclear. To investigate whether TGFβ signaling might be disrupted by inactivation of the key signaling molecule, the TGFβ type I (TβR‐I) receptor, and whether or not TβR‐I inactivation is associated with late stage disease, we conducted a comprehensive structural analysis of the TβR‐I gene in fine‐needle aspirates of 23 head‐&‐neck cancer metastases. We encountered 4 different mutations of TβR‐I, 3 of which have not been previously identified. In 1 case, we found a somatic intragenic 4‐bp deletion predicting for a truncation of the receptor protein. This is the first example of a true loss‐of‐function mutation of TβR‐I in a human epithelial neoplasm. In 2 other cases, we identified missense mutations located between the juxtamembrane‐ and serine‐threonine kinase domains. One of these resulted in an alanine‐to‐threonine substitution (A230T), which disrupts receptor signaling activity by causing rapid protein degradation within the endoplasmatic reticulum. This represents a novel mechanism of inactivation of a TGFβ signaling intermediate. Finally, we identified a serine‐to‐tyrosine substitution at codon 387 (S387Y) in a metastasis but not in the corresponding primary tumor. We had previously shown this S387Y mutant to be predominantly associated with breast cancer metastases and to have a diminished ability to mediate TGFβ‐dependent signaling. In aggregate, these findings provide further support for the hypothesis that inactivation of the TGFβ signaling pathway occurs in a significant subset of human cancers.

Breast cancer prognosis by combinatorial analysis of gene expression data

IntroductionThe potential of applying data analysis tools to microarray data for diagnosis and prognosis is illustrated on the recent breast cancer dataset of van t Veer and coworkers. We re-examine that dataset using the novel technique of logical analysis of data (LAD), with the double objective of discovering patterns characteristic for cases with good or poor outcome, using them for accurate and justifiable predictions; and deriving novel information about the role of genes, the existence of special classes of cases, and other factors.MethodData were analyzed using the combinatorics and optimization-based method of LAD, recently shown to provide highly accurate diagnostic and prognostic systems in cardiology, cancer proteomics, hematology, pulmonology, and other disciplines.ResultsLAD identified a subset of 17 of the 25,000 genes, capable of fully distinguishing between patients with poor, respectively good prognoses. An extensive list of patterns or combinatorial biomarkers (that is, combinations of genes and limitations on their expression levels) was generated, and 40 patterns were used to create a prognostic system, shown to have 100% and 92.9% weighted accuracy on the training and test sets, respectively. The prognostic system uses fewer genes than other methods, and has similar or better accuracy than those reported in other studies. Out of the 17 genes identified by LAD, three (respectively, five) were shown to play a significant role in determining poor (respectively, good) prognosis. Two new classes of patients (described by similar sets of covering patterns, gene expression ranges, and clinical features) were discovered. As a by-product of the study, it is shown that the training and the test sets of van t Veer have differing characteristics.ConclusionThe study shows that LAD provides an accurate and fully explanatory prognostic system for breast cancer using genomic data (that is, a system that, in addition to predicting good or poor prognosis, provides an individualized explanation of the reasons for that prognosis for each patient). Moreover, the LAD model provides valuable insights into the roles of individual and combinatorial biomarkers, allows the discovery of new classes of patients, and generates a vast library of biomedical research hypotheses.

A prototype for unsupervised analysis of tissue microarrays for cancer research and diagnostics

The tissue microarray (TMA) technique enables researchers to extract small cylinders of tissue from histological sections and arrange them in a matrix configuration on a recipient paraffin block such that hundreds can be analyzed simultaneously. TMA offers several advantages over traditional specimen preparation by maximizing limited tissue resources and providing a highly efficient means for visualizing molecular targets. By enabling researchers to reliably determine the protein expression profile for specific types of cancer, it may be possible to elucidate the mechanism by which healthy tissues are transformed into malignancies. Currently, the primary methods used to evaluate arrays involve the interactive review of TMA samples while they are viewed under a microscope, subjectively evaluated, and scored by a technician. This process is extremely slow, tedious, and prone to error. In order to facilitate large-scale, multi-institutional studies, a more automated and reliable means for analyzing TMAs is needed. We report here a web-based prototype which features automated imaging, registration, and distributed archiving of TMAs in multiuser network environments. The system utilizes a principal color decomposition approach to identify and characterize the predominant staining signatures of specimens in color space. This strategy was shown to be reliable for detecting and quantifying the immunohistochemical expression levels for TMAs.

Alterations of Smad expression and activation in defining 2 subtypes of human head and neck squamous cell carcinoma

We postulated that disruptions of the canonical transforming growth factor‐beta (TGF‐β)/Smad signaling pathway might contribute to the development of head and neck squamous cell carcinoma (HNSCC).