Richard L. Klemke

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

Spatiotemporal dynamics of RhoA activity in migrating cells

Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body. However, there is evidence that RhoA also regulates membrane protrusion. Here we use a fluorescent biosensor, based on a novel design preserving reversible membrane interactions, to visualize the spatiotemporal dynamics of RhoA activity during cell migration. In randomly migrating cells, RhoA activity is concentrated in a sharp band directly at the edge of protrusions. It is observed sporadically in retracting tails, and is low in the cell body. RhoA activity is also associated with peripheral ruffles and pinocytic vesicles, but not with dorsal ruffles induced by platelet-derived growth factor (PDGF). In contrast to randomly migrating cells, PDGF-induced membrane protrusions have low RhoA activity, potentially because PDGF strongly activates Rac, which has previously been shown to antagonize RhoA activity. Our data therefore show that different extracellular cues induce distinct patterns of RhoA signalling during membrane protrusion.

Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells

The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20–35% changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells.

Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells†

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed‐phase liquid chromatography strategy as a first dimension for two‐dimensional liquid chromatography tandem mass spectrometry (“shotgun”) proteomic analysis of trypsin‐digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed‐phase liquid chromatography as a first‐dimension fractionation strategy resulted in 1.8‐ and 1.6‐fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed‐phased strategy is an attractive alternative to strong cation exchange for two‐dimensional shotgun proteomic analysis.

Four Human Ras Homologs Differ in Their Abilities to Activate Raf-1, Induce Transformation, and Stimulate Cell Motility

Human cells contain four homologous Ras proteins, but it is unknown whether each of these Ras proteins participates in distinct signal transduction cascades or has different biological functions. To directly address these issues, we assessed the relative ability of constitutively active (G12V) versions of each of the four Ras homologs to activate the effector protein Raf-1 in vivo. In addition, we compared their relative abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell migration. We found a distinct hierarchy between the four Ras homologs in each of the parameters studied. The hierarchies were as follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>> N-Ras > Ha-Ras; for focus formation, Ha-Ras ≥ Ki-Ras 4A >>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras 4A ≥ N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras 4A = no migration. Our results indicate that the four Ras homologs significantly differ in their abilities to activate Raf-1 and induce distinctly different biological responses. These studies, in conjunction with our previous report that demonstrated that the Ras homologs can be differentially activated by upstream guanine nucleotide exchange factors (Jones, M. K., and Jackson, J. H. (1998)J. Biol. Chem. 273, 1782–1787), indicate that each of the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also suggest for the first time that the distinct and likely cooperative biological functions of the Ki-ras-encoded Ki-Ras 4A and Ki-Ras 4B proteins may help explain why constitutively activating mutations of Ki-ras, but not N-ras or Ha-ras, are frequently detected in human carcinomas.

High-resolution imaging of the dynamic tumor cell–vascular interface in transparent zebrafish

Cell metastasis is a highly dynamic process that occurs in multiple steps. Understanding this process has been limited by the inability to visualize tumor cell behavior in real time by using animal models. Here, we employ translucent zebrafish and high-resolution confocal microscopy to study how human cancer cells invade in tissues, induce angiogenesis, and interact with newly formed vessels. We use this system to study how the human metastatic gene RhoC promotes the initial steps of metastasis. We find that RhoC expression induces a primitive amoeboid-like cell invasion characterized by the formation of dynamic membrane protrusions and blebs. Surprisingly, these structures penetrate the blood vessel wall exclusively at sites of vascular remodeling and not at regions of existing intact vessels. This process requires tumor cells to secrete VEGF, which induces vascular openings, which in turn, serve as portholes allowing access of RhoC-expressing cells to the blood system. Our results support a model in which the early steps in intravasation and metastasis require two independent events: (i) dynamic regulation of the actin/myosin cytoskeleton within the tumor cell to form protrusive structures and (ii) vascular permeablization and vessel remodeling. The integration of zebrafish transgenic technology with human cancer biology may aid in the development of cancer models that target specific organs, tissues, or cell types within the tumors. Zebrafish could also provide a cost-effective means for the rapid development of therapeutic agents directed at blocking human cancer progression and tumor-induced angiogenesis.

Visualizing extravasation dynamics of metastatic tumor cells.

Little is known about how metastatic cancer cells arrest in small capillaries and traverse the vascular wall during extravasation in vivo. Using real-time intravital imaging of human tumor cells transplanted into transparent zebrafish, we show here that extravasation of cancer cells is a highly dynamic process that involves the modulation of tumor cell adhesion to the endothelium and intravascular cell migration along the luminal surface of the vascular wall. Tumor cells do not damage or induce vascular leak at the site of extravasation, but rather induce local vessel remodeling characterized by clustering of endothelial cells and cell-cell junctions. Intravascular locomotion of tumor cells is independent of the direction of blood flow and requires β1-integrin-mediated adhesion to the blood-vessel wall. Interestingly, the expression of the pro-metastatic gene Twist in tumor cells increases their intravascular migration and extravasation through the vessel wall. However, in this case, Twist expression causes the tumor cells to switch to a β1-integrin-independent mode of extravasation that is associated with the formation of large dynamic rounded membrane protrusions. Our results demonstrate that extravasation of tumor cells is a highly dynamic process influenced by metastatic genes that target adhesion and intravascular migration of tumor cells, and induce endothelial remodeling.

Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold.

Initiation of cell migration requires morphological polarization with formation of a dominant leading pseudopodium and rear compartment. A molecular understanding of this process has been limited, due to the inability to biochemically separate the leading pseudopodium from the rear of the cell. Here we examine the spatio-temporal localization and activation of cytoskeletal-associated signals in purified pseudopodia directed to undergo growth or retraction. Pseudopodia growth requires assembly of a p130Crk-associated substrate (CAS)/c-CrkII (Crk) scaffold, which facilitates translocation and activation of Rac1. Interestingly, Rac1 activation then serves as a positive-feedback loop to maintain CAS/Crk coupling and pseudopodia extension. Conversely, disassembly of this molecular scaffold is critical for export and down regulation of Rac1 activity and induction of pseudopodia retraction. Surprisingly, the uncoupling of Crk from CAS during pseudopodium retraction is independent of changes in focal adhesion kinase activity and CAS tyrosine phosphorylation. These findings establish CAS/Crk as an essential scaffold for Rac1-mediated pseudopodia growth and retraction, and illustrate spatio-temporal segregation of cytoskeletal signals during cell polarization.

Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo.

Tumor cell interactions with adhesion proteins and growth factors likely contribute to the metastatic cascade. Evidence is provided that insulin or insulin-like growth factor-mediated signals cooperate with the commonly expressed integrin alpha v beta 5 to promote spontaneous pulmonary metastasis of multiple tumor cell types in both the chick embryo and severe combined immune deficiency mouse/human chimeric models. Expression of alpha v beta 5 in tumor cells promoted their adhesion to vitronectin in vitro. However, cell motility required cytokine stimulation, which caused redistribution of alpha-actinin to membrane-adhesive sites containing alpha v beta 5. Significantly, ligation of alpha v beta 5 and cytokine receptors were both required for spontaneous pulmonary metastasis of multiple tumor types even though it was not necessary for primary tumor growth. Thus, tumor cell metastasis can be regulated by a functional cooperation between cytokine signaling events and the adhesion receptor alpha v beta 5 in a manner independent of tumor cell growth. These findings provide evidence that integrin ligation, in conjunction with cytokine activation, plays an important role in the dissemination of malignant tumor cells.

Catch of the day: zebrafish as a human cancer model

Zebrafish are making big waves in the field of cancer research. The effect has been widespread and continues to gain speed as more and more cancer researchers ride the wave of zebrafish biology. This has been largely due to the development of transgenic and xenograft models of cancer, which recapitulate many aspects of different human cancers including lymphoblastic T-cell leukemia, pancreatic cancer, melanoma and rhabdomyosarcoma. These models are already being utilized by academia and industry to search for genetic and chemical modifiers of cancer with success. The attention has been further stimulated by the amenability of zebrafish to pharmacological testing and the superior imaging properties of fish tissues that allow visualization of cancer progression and angiogenesis in live animals. This review summarizes the current zebrafish models of cancer and discusses their utility in human cancer research and future directions in the field.