Analysis of acquired resistance to metronomic oral topotecan chemotherapy plus pazopanib after prolonged preclinical potent responsiveness in advanced ovarian cancer.

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy--the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. the anti-VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.

The crystal structure and dimerization interface of GADD45gamma.

Gadd45 proteins are recognized as tumor and autoimmune suppressors whose expression can be induced by genotoxic stresses. These proteins are involved in cell cycle control, growth arrest, and apoptosis through interactions with a wide variety of binding partners. We report here the crystal structure of Gadd45gamma, which reveals a fold comprising an alphabetaalpha sandwich with a central five-stranded mixed beta-sheet with alpha-helices packed on either side. Based on crystallographic symmetry we identified the dimer interface of Gadd45gamma dimers by generating point mutants that compromised dimerization while leaving the tertiary structure of the monomer intact. The dimer interface comprises a four-helix bundle involving residues that are the most highly conserved among Gadd45 isoforms. Cell-based assays using these point mutants demonstrate that dimerization is essential for growth inhibition. This structural information provides a new context for evaluation of the plethora of protein-protein interactions that govern the many functions of the Gadd45 family of proteins.

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Signaling network assessment of mutations and copy number variations predict breast cancer subtype-specific drug targets.

Individual cancer cells carry a bewildering number of distinct genomic alterations (e.g., copy number variations and mutations), making it a challenge to uncover genomic-driven mechanisms governing tumorigenesis. Here, we performed exome sequencing on several breast cancer cell lines that represent two subtypes, luminal and basal. We integrated these sequencing data and functional RNAi screening data (for the identification of genes that are essential for cell proliferation and survival) onto a human signaling network. Two subtype-specific networks that potentially represent core-signaling mechanisms underlying tumorigenesis were identified. Within both networks, we found that genes were differentially affected in different cell lines; i.e., in some cell lines a gene was identified through RNAi screening, whereas in others it was genomically altered. Interestingly, we found that highly connected network genes could be used to correctly classify breast tumors into subtypes on the basis of genomic alterations. Further, the networks effectively predicted subtype-specific drug targets, which were experimentally validated.

Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma.

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases.

Differential sensitivity of A549 non-small lung carcinoma cell responses to epidermal growth factor receptor pathway inhibitors.

It has been demonstrated that A549 non-small cell lung cancer (NSCLC) cells are sensitive to epidermal growth factor receptor (EGFR) inhibitors in in vivo xenograft animal models, but are relatively resistant in conventional in vitro monolayer growth assays. Here, we utilized anchorage-independent cell growth/survival assays as well as motility assays and demonstrated that these tests detect the effects of two EGFR inhibitors, the small molecule inhibitor AG1478 and the ligand-blocking antibody 225 mAb, on A549 cells more sensitively than monolayer growth assays. AG1478 was more effective than 225 mAb at inhibiting EGF-stimulated anchorage-independent cell growth, in part due to its pronounced ability to inhibit cell survival, whereas 225 mAb and AG1478 were both able to inhibit cell motility. In order to determine which EGFR signalling pathway components were most strongly associated with these cell responses, we analyzed in parallel the phosphorylation levels of EGFR itself as well as several downstream pathway elements. We found that the limited ability of 225 mAb to inhibit MAPK, PI3K and STAT3 phosphorylation correlated with its inability to promote anchorage independent apoptosis, but did not correlate with its ability to inhibit motility. Based on our results in A549 cells, we propose that EGF stimulates tumour progression of NSCLC largely through effects on anchorage-independent growth and survival, as well as motility.