Overproduction of IGF-2 drives a subset of colorectal cancer cells, which specifically respond to an anti-IGF therapeutic antibody and combination therapies.

Colorectal cancer (CRC) is a heterogeneous disease with a broad spectrum of genetic and epigenetic changes. A comprehensive molecular characterization of CRC by The Cancer Genome Atlas Network detected the overexpression of the insulin-like growth factor 2 (IGF2) gene, encoding a ligand for the insulin-like growth factor 1 receptor (IGF-1R), in a subset of CRC tumors. In this study, we investigated the oncogenic potential of IGF-2 in IGF2-overexpressing CRC models and the efficacy of MEDI-573, an IGF-1/2-neutralizing antibody. We found that a subset of CRC cell lines express high IGF-2 levels owing to an increased DNA copy number and hypermethylation in the H19 promoter of the IGF2 gene. MEDI-573 efficiently neutralized IGF-2 and induced apoptosis, which resulted in significant tumor growth inhibition in CRC mouse models that express high levels of IGF-2. These effects were specific to CRCs overexpressing IGF-2, as MEDI-573 did not affect the growth CRC cell lines with normal levels. Moreover, blockade of IGF-2 by MEDI-573 modulated other signaling pathways, suggesting combination therapies with inhibitors of these pathways. Indeed, in vivo efficacy was significantly enhanced when MEDI-573 was used in combination with trastuzumab, AZD2014 (dual mTORC1/2i), AZD5363 (AKTi) and selumetinib (AZD6244/ARRY-142886, MEK1/2i) or cetuximab. These results demonstrate that overexpressed IGF-2 is the major tumorigenic driver in a subset of CRCs and encourage testing of MEDI-573, alone and in combinations, in IGF2-overexpressing CRC patients.

HER2 drives Mucin-like 1 to control proliferation in breast cancer cells.

Mucin-like 1 (MUCL1) was first identified as a breast-specific gene over a decade ago. Based on its highly restricted mRNA expression in breast tissue and continued expression during breast tumorigenesis and progression, MUCL1 is an attractive tumor-associated antigen and a potential therapeutic target. However, very little is known about the cellular location, biological functions and regulation of the MUCL1 protein, which will have a major impact on its druggability. Here we describe our efforts to fully characterize the cellular localization of MUCL1, investigate its regulation by key breast cancer oncogenes such as human epidermal growth factor receptor 2 (HER2) and discover its functional roles in breast cancer. Although some mucins are membrane bound, our data indicate that MUCL1 is secreted by some breast cancer cells, whereas others only express high levels of intracellular MUCL1. MUCL1 expression is highest in HER2-amplified breast tumors and inhibiting HER2 activity in tumor cells resulted in a decreased MUCL1 expression. In-depth investigation demonstrated that phosphoinositide3-kinase/Akt pathway, but not Ras/MEK pathway, controls MUCL1 expression downstream of HER2. Phenotypic assays revealed a strong dependence of HER2-positive cells on MUCL1 for cell proliferation. We further identified the mechanism by which MUCL1 regulates cell growth. Knockdown of MUCL1 induced a G1/S phase arrest concomitant with decreased cyclin D and increased p21 and p27 levels. Finally, we investigated the impact of MUCL1 loss on kinase signaling pathways in breast cancer cells through phospho-kinase array profiling. MUCL1 silencing abrogated phospho-focal adhesion kinase (FAK), Jun NH2-terminal kinase (JNK) and c-Jun signals, but not extracellular signal-regulated kinase or Akt pathway activities, thereby pointing to FAK/JNK pathway as the downstream effector of MUCL1 signaling. We are the first to identify an important role for MUCL1 in the proliferation of breast cancer cells, probably mediated via the FAK/JNK signaling pathway. Taken together, these data suggest a potential utility for therapeutic targeting of this protein in breast cancer.

Spiral plasmonic nanoantennas as circular polarization transmission filters.

We present simulation and experimental results for easily fabricated spiral plasmonic antenna analogues providing circular polarization selectivity. One circular polarization state is concentrated and transmitted through a subwavelength aperture, while the opposite circular state is blocked. The spectral bandwidth, efficiency, and extinction ratios are tunable through geometric parameters. Integration of such structures onto a focal plane array in conjunction with linear micropolarizers enables complete Stokes vector imaging, that, until now, has been difficult to achieve. An array of these structures forms a plasmonic metamaterial that exhibits high circular dichroism.

Ultra-high extinction ratio micropolarizers using plasmonic lenses.

The design of a new type of plasmonic ultra-high extinction ratio micropolarizing transmission filter is presented along with an experimental demonstration. A pair of dielectric coated metal gratings couple incident TM polarized light into surface plasmons, which are fed into a central metal-insulator-metal (MIM) waveguide, followed by transmission through a sub-wavelength aperture. Extinction ratios exceeding 10¹¹ are predicted by finite element simulation. Good absolute agreement for both the spectral and polarization response is obtained between measurement and simulations using measured geometric parameters. The filters can be easily fabricated and sized to match the pixel pitch of current focal plane arrays.

Hybrid plasmon/dielectric waveguide for integrated silicon-on-insulator optical elements.

VLSI compatible optical waveguides on silicon are currently of particular interest in order to integrate optical elements onto silicon chips, and for possible replacements of electrical cross-chip/inter-core interconnects. Here we present simulation and experimental verification of a hybrid plasmon/dielectric, single-mode, single-polarization waveguide for silicon-on-insulator wafers. Its fabrication is compatible with VLSI processing techniques, and it possesses desirable properties such as the absence of birefringence and low sensitivity to surface roughness and metallic losses. The waveguide structure naturally forms an MOS capacitor, possibly useful for active device integration. Simulations predict very long propagation lengths of millimeter scale with micron scale confinement, or sub-micron scale confinement with propagation lengths still in excess of 100 microns. The waveguide may be tuned continuously between these states using standard VLSI processing. Extremely long propagation lengths have been simulated: one configuration presented here has a simulated propagation length of 34 cm.

Interference and resonant cavity effects explain enhanced transmission through subwavelength apertures in thin metal films.

Transmission through an opaque Au film with a single subwavelength aperture centered in a smooth cavity between linear grating structures is studied experimentally and with a finite element model. The model is in good agreement with measured results and is used to investigate local field behavior. It shows that a surface plasmon polariton (SPP) is launched along the metal surface, while interference of the SPP with the incident light along with resonant cavity effects give rise to suppression and enhancement in transmission. Based on experimental and modeling results, peak location and structure of the enhancement/suppression bands are explained analytically, confirming the primary role of SPPs in enhanced transmission through small apertures in opaque metal films.

PTCH2, a novel human patched gene, undergoing alternative splicing and up-regulated in basal cell carcinomas.

By a combination of cDNA library screening, rapid amplification of cDNA ends analysis, and BAC sequencing, a novel human patched-like gene (PTCH2) has been cloned and sequenced. The genomic organization is similar to PTCH1 with 22 exons and, by radiation hybrid mapping, PTCH2 has been localized to chromosome 1p33-34, a region often lost in a variety of tumors. Several alternatively spliced mRNA forms of PTCH2 were identified, including transcripts lacking segments thought to be involved in sonic hedgehog binding and mRNAs with differentially defined 3' terminal exons. In situ hybridization revealed high expression of PTCH2 transcripts in both familial and sporadic basal cell carcinomas in similarity to what has been observed for PTCH1, suggesting a negative regulation of PTCH2 by PTCH1. This finding tightly links PTCH2 with the sonic hedgehog/PTCH signaling pathway, implying that PTCH2 has related, but yet distinct, functions than PTCH1.

A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines.

The Cdc7 protein kinase of Saccharomyces cerevisiae is a critical regulator of several aspects of DNA metabolism and cell cycle progression. We describe the isolation of a human gene encoding a Cdc7 homolog. The Cdc7Hs protein sequence is 27% identical to that of the yeast protein, includes features unique to yeast Cdc7, and contains all conserved catalytic residues of protein kinases. The human sequence also shows significant similarity to the cyclin-dependent kinases, in accordance with evidence that yeast Cdc7 is related to the cdks. CDC7Hs is expressed in many normal tissues, but overexpressed in certain tumor types and all transformed cell lines examined. In some of the tumors tested, CDC7Hs expression correlates with expression of a proliferation marker, the histone H3 gene. In other cases, no such correlation was observed. This suggests that CDC7Hs expression may be associated hyperproliferation in some tumors and neoplastic transformation in others.

A retinoblastoma-binding protein related to a negative regulator of Ras in yeast.

The growth suppression function of the retinoblastoma protein (Rb) is though to be mediated by Rb binding to cellular proteins. p48 is one of the major proteins that binds to a putative functional domain at the carboxy terminus of the Rb protein. Here we report the isolation of a full-length complementary DNA (RbAp48) encoding p48. Complex formation between p48 and Rb occurs in vitro and in vivo, and apparently involves direct interaction between the proteins. Like Rb, p48 is a ubiquitously expressed nuclear protein. RbAp48 share sequence homology with MSI1, a negative regulator of the Ras-cyclic AMP pathway in the yeast Saccharomyces cerevisiae. Furthermore, like MSI1, human RbAp48 suppresses the heat-shock sensitivity of the yeast ira1 strains and RAS2Val19 strains. Interaction with p48 may be one of the mechanisms for suppression of growth mediated by Rb.

Yeast pre-meiotic DNA replication utilizes mitotic origin ARS1 independently of CDC7 function.

In budding yeast, mitotic DNA replication initiates at sequence-specific replication origins, the prototype for which is ARS1. Initiation serves as the primary control point for mitotic DNA replication, and is catalyzed by the Cdc7 protein kinase. In contrast, premeiotic DNA replication apparently does not require Cdc7, and the existence and nature of specific replication origins in the meiotic division cycle have not been previously reported. We have begun to investigate the mechanism of premeiotic DNA synthesis by determining whether or not ARS1 functions as a DNA replication origin in meiosis. We have taken advantage of the fact that transcription through ARS1 disrupts its ability to function as an origin to show that ARS1 is required for premeiotic DNA replication of a plasmid bearing this element. Further, premeiotic replication from ARS1 still occurs in a cdc7 mutant strain held at conditions non-permissive for Cdc7 protein kinase activity. These findings reveal that premeiotic DNA replication can initiate from origins also used in mitosis, and is not regulated by Cdc7. Taken together with previous findings implicating Cdc7 in meiotic DNA recombination and induced mutagenesis, these findings prompt us to postulate that the Cdc7 protein kinase regulates some step common to several DNA metabolic processes such as local disassembly of chromatin or activation of a key component of the DNA metabolic machinery.

Integration of cell cycle control with transcriptional regulation by the retinoblastoma protein.

Rapid progress in several areas of molecular biology has led to the realization that the retinoblastoma protein may play a pivotal role in the coordination between cell cycle control and regulation of gene expression. This role is a subtle one, and is important in only certain mammalian cell types. Exploring the details of these connections, and why only some cells rely on them, is already beginning to shed light on the regulation of cell multiplication.

Retinoblastoma protein and the cell cycle.

Deregulation of the cell cycle may contribute one of the primary mechanisms through which cancer arises. Eukaryotic cell division has been found to be a strictly controlled process, involving response to both positive and negative external signals and assessment of the cell's internal state. Several recent discoveries have strengthened and refined the theory that the retinoblastoma protein is involved in the decision between cell division and differentiation, and have begun to provide an outline of the nature of this involvement.

Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast.

The Saccharomyces cerevisiae CDC7 gene encodes a protein kinase that functions in DNA replication, repair, and meiotic recombination. The sequence of several temperature-sensitive (ts) cdc7 mutations was determined and correlated with protein kinase consensus domain structure. The positions of these ts alleles suggests some general principles for predicting ts protein kinase mutations. Pedigree segregation lag analysis demonstrated that all of the mutant proteins are less active or less stable than wild-type Cdc7p. Two new mutations were constructed, one by site-directed and the other by insertional mutagenesis. All of the cdc7 mutants were assayed for induced mutagenesis in response to mutagenic agents at the permissive temperature. Some cdc7 mutants were found to be hypomutable, while others are hypermutable. The differences in mutability are observed most clearly when log phase cells are used. Both hypo- and hypermutability are recessive to wild type. Cdc7p may participate in DNA repair by phosphorylating repair enzymes or by altering chromatin structure to allow accessibility to DNA lesions.

Tumor suppressor genes: new prospects for cancer research.

Cancer is thought to arise from the accumulation of several genetic mutations in a single cell. Until recently, the only tumorigenic mutations that have been studied in detail are those that activate oncogenes. The discovery of tumor suppressor genes, for which inactivating mutations elicit tumorigenesis, has added a new dimension to our understanding of neoplasia. The retinoblastoma susceptibility gene RB is the prototype tumor suppressor gene and has been shown to suppress the transformed phenotype for several different cancers. Additional studies have revealed other tumor suppressor genes that may operate in a variety of tissues through a variety of mechanisms. These mechanisms may regulate the choice between cellular proliferation and differentiation and appear to involve such processes as the initiation of DNA replication, regulation of expression of certain genes, intercellular communication and adhesion, and the transduction of external signals to intracellular effectors. The elucidation of these mechanisms will enhance our understanding of both oncogenesis and the fundamental operations of the cell.

DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase.

The yeast Cdc7 protein is indispensable to initiation of nuclear DNA replication, based on the phenotype of the conditional, temperature-sensitive (ts) cdc7 mutants at the restrictive temperature. This protein has likewise been implicated in commitment to meiotic DNA recombination and induced mutagenesis, which may result from error-prone DNA repair. Our previous work revealed sequence similarity between the Cdc7 protein and known protein kinases. To determine whether it possesses kinase activity, we have immunoprecipitated the protein from Cdc7-overproducing yeast cells by using polyclonal antibodies raised against a nondenatured beta-galactosidase-Cdc7 fusion protein. In this report, we demonstrate that Cdc7 immune complexes are capable of phosphorylating mammalian histone H1 on serine and/or threonine residues. Immune complexes derived from cells harboring the cdc7-2 ts mutant gene on a high copy number plasmid possess a thermolabile kinase activity. Thus, we postulate that Cdc7 may regulate the various DNA metabolic pathways by phosphorylating one or more target substrates. Because Cdc7 kinase acts downstream of Cdc28/cdc2 kinase function at "start," the transition from G1 to S phase in the cell cycle may be the result of a cascade of protein phosphorylation.