In vitro and in vivo studies of the combination of IGF1R inhibitor figitumumab (CP-751,871) with HER2 inhibitors trastuzumab and neratinib.

The insulin-like growth factor I receptor (IGF1R) has been linked to resistance to HER2-directed therapy with trastuzumab (Herceptin). We examined the anti-tumor activity of figitumumab (CP-751,871), a human monoclonal antibody that blocks IGF1R ligand binding, alone and in combination with the therapeutic anti-HER2 antibody trastuzumab and the pan-HER family tyrosine kinase inhibitor neratinib, using in vitro and in vivo breast cancer model systems. In vitro assays of proliferation, apoptosis, and signaling, and in vivo anti-tumor experiments were conducted in HER2-overexpressing (BT474) and HER2-normal (MCF7) models. We find single-agent activity of the HER2-targeting drugs but not figitumumab in the BT474 model, while the reverse is true in the MCF7 model. However, in both models, combining figitumumab with HER2-targeting drugs shows synergistic anti-proliferative and apoptosis-inducing effects, and optimum inhibition of downstream signaling. In murine xenograft models, synergistic anti-tumor effects were observed in the HER2-normal MCF7 model for the combination of figitumumab with trastuzumab, and, in the HER2-overexpressing BT474 model, enhanced anti-tumor effects were observed for the combination of figitumumab with either trastuzumab or neratinib. Analysis of tumor extracts from the in vivo experiments showed evidence of the most optimal inhibition of downstream signaling for the drug combinations over the single-agent therapies. These results suggest promise for such combinations in treating patients with breast cancer, and that, unlike the case for single-agent therapy, the therapeutic effects of such combinations may be independent of expression levels of the individual receptors or the single-agent activity profile.

Co-targeting the insulin-like growth factor I receptor enhances growth-inhibitory and pro-apoptotic effects of anti-estrogens in human breast cancer cell lines.

The insulin-like growth factor I receptor (IGF1R) interacts with estrogen receptor-alpha (ERalpha) and HER2. We examined the effect of combinations of IGF1R antagonists (alpha-IR3, AG1024) and anti-estrogens (4-hydroxy tamoxifen, fulvestrant) in two human ER+ breast cancer cell lines: BT474 (HER2 overexpressing, IGF1R low) and MCF7 (HER2 non-overexpressing, IGF1R high). In BT474 cells, growth was inhibited by anti-estrogens, but not by IGF1R antagonists; however, adding IGF1R inhibitors to anti-estrogens enhanced growth inhibition. In MCF7 cells, growth was inhibited by IGF1R and ER antagonists and more so by their combination. In both cell lines, no single agents could induce apoptosis, but combining IGF1R inhibitors with anti-estrogens induced dramatic levels of apoptosis. IGF1R antagonists enhanced the ability of the anti-estrogens to inhibit ER transcriptional activity in BT474 cells, but not in MCF7 cells. The drug combination synergistically inhibited ER and IGF1R activity. Such combinations may be useful therapy for breast cancer.

The cancer cell--leukocyte fusion theory of metastasis.

The cause of metastasis remains elusive despite vast information on cancer cells. We posit that cancer cell fusion with macrophages or other migratory bone marrow-derived cells (BMDCs) provides an explanation. BMDCs fused with tumor cells were present in animal tumor xenografts where they were associated with metastases. In myeloma patients, transcriptionally active myeloma nuclei were incorporated into osteoclasts through fusion. In patients with renal cell carcinoma arising poststem cell transplant, donor genes were incorporated in recipient cancer cell nuclei, most likely through fusion, and showed tumor distribution patterns characteristic of cancer stem cells. Melanoma-macrophage hybrids generated in vitro contained chromosomes from both parental partners, showed increased ploidy, and transcribed and translated genes from both parents. They exhibited chemotactic migration in vitro toward fibronectin and exhibited high frequencies of metastasis when implanted in mice. They produced macromolecules that are characteristic of macrophages and known indicators of metastasis (c-Met, SPARC, MCR1, GnT-V, and the integrin subunits alpha(3), alpha(5), alpha(6), alpha(v), beta(1), beta(3)). They also produced high levels of beta1,6-branched oligosaccharides-predictors of poor survival in patients with melanoma or carcinomas of the breast, lung, and colon. We thus hypothesize that such gene expression patterns in cancer are generated through fusion. Tumor hybrids also showed active autophagy, a characteristic of both metastatic cancers and macrophages. BMDC-tumor cell fusion explains epidermal-mesenchymal transition in cancer since BMDCs express mesodermal traits and epithelial-mesenchymal transition regulators (Twist, SPARC, and others). If BMDC-tumor cell fusion underlies invasion and metastasis in human cancer, new approaches for therapeutic intervention would be mandated.

Evidence for tyrosinase as a beta1,6 branch containing glycoprotein: substrate of GnT-V.

Tyrosinase is a rate-limiting enzyme in mammalian melanogenesis, and is known as a glycoprotein. Post-translational processing of mammalian tyrosinase is required for its folding, sorting, and for enzymatic activity. Here we show for the first time that the mammalian tyrosinase has beta1,6-branched N-glycan structure that can be recognized by binding with specific lectin Leukoagglutinating phytohematoagglutinin (L-PHA). Further, this specific glycoconjugate structure has been shown to have a function relationship in melanin synthesis.

Beta1,6-branched oligosaccharides regulate melanin content and motility in macrophage-melanoma fusion hybrids.

In previous studies, fusion of peritoneal macrophages or blood monocytes with mouse melanoma cells produced hybrids with upregulated expression of the glycosyltransferase beta1,6-N-acetylglucosaminyltransferase V (GnT-V) and its enzymatic product, beta1,6-branched oligosaccharides. This correlated with marked increases in motility, metastatic potential and, surprisingly, melanin content. This study was designed to establish direct roles for beta1,6-branched oligosaccharides in melanogenesis and motility. The levels of beta1,6-branched oligosaccharides were lowered by transfecting beta1,4-N-acetylglucosaminyltransferase III, a competitive inhibitor of GnT-V. beta1,4-N-acetylglucosaminyltransferase III transfection virtually eliminated melanin production and markedly decreased chemotactic motility. This implied that the metastatic and melanogenic phenotypes in hybrids were each upregulated by beta1,6-branched oligosaccharides. Although roles for beta1,6-branched oligosaccharides in motility and metastasis have been reported previously, this is the first study to directly implicate these structures in melanogenesis. Although drawn from experimental models, the findings might explain the well known hypermelanotic regions of human cutaneous malignant melanoma as hypermelanotic cutaneous malignant melanoma cells are rich in beta1,6-branched oligosaccharides. They might also explain why melanogenesis pathways differ between malignant and normal melanocytes as GnT-V is a myeloid-associated enzyme that is aberrantly expressed in melanoma cells but not in normal melanocytes.

GnT-V expression and metastatic phenotypes in macrophage-melanoma fusion hybrids is down-regulated by 5-Aza-dC: evidence for methylation sensitive, extragenic regulation of GnT-V transcription.

Beta1,6-acetylglucosaminyltransferase V (GnT-V) forms beta1,6 branching on the trimannosyl terminus of N-glycans, allowing for the production of beta1,6Glc-NAc-bearing oligosaccharides. These are used by healthy myeloid cells and cancer cells alike for systemic migration. GnT-V has multiple glycoprotein substrates and thereby exerts global effects on cancer progression, characteristic of a master regulator of metastasis. Yet little is known of the regulation of GnT-V expression by tumor cells. It was previously reported that fusion of macrophages with Cloudman S91 mouse melanoma cells produced macrophage-melanoma hybrids with up-regulated GnT-V expression regarding mRNA and enzymatic activity. Majority of these hybrids showed increased chemotactic motility in vitro and elevated metastatic potential in vivo. Here we attempted to understand this at the molecular genetic level focusing on DNA hypermethylation as a potentially key step. Treatment of cells with 5-Aza-dC, an inhibitor of DNA methylation, resulted in decreased expression of GnT-V mRNA and beta1,6-branched oligosaccharides along with reduced glycosylation of LAMP-1, a major substrate for GnT-V. This was accompanied by reduced chemotactic motility of the cells. The results suggested that DNA hypermethylation in some fashion stimulated GnT-V expression. We thus investigated the promoter region of the GnT-V gene for hypermethylation of CpG islands, comparing macrophage-melanoma hybrids of low and high metastatic potential with the parental melanoma cell line. Genomic DNA after bisulfite modification amplified from this region showed identical sequences between the cell lines. The findings indicated that differential methylation of the promoter region of GnT-V gene was not responsible for its transcriptional control, rather, appeared to be controlled through a negative regulator, nm23, whose own expression was regulated by hypermethylation. Although our studies involved a highly experimental system, the results further suggest that by whatever mechanism, reduction of GnT-V activity through 5-Aza-dC treatment might provide a new approach towards prevention of metastatic progression.

Co-targeting ER and HER family receptors induces apoptosis in HER2-normal or overexpressing breast cancer models.

BACKGROUND: Estrogen receptor (ER) and human epidermal growth factor receptor (HER) family receptors interact in breast cancer; co-targeting these receptors is of interest. We previously reported on a synergistic growth inhibition for the combination of trastuzumab plus tamoxifen in HER2+/ER+ BT474 cells, but no induction of apoptosis. Herein we describe the effects of co-targeting in models of differing HER2 overexpression status (MCF7 HER2-normal/ER+, BT474 HER2-overexpressing/ER+). MATERIALS AND METHODS: Assays of proliferation were carried-out using WST-1, cell cycle using flow cytometry, and apoptosis by determination of sub-G1 population and by annexin-V. RESULTS: Combining a dual HER2/EGFR kinase inhibitor with anti-estrogens induces apoptosis of BT474 cells. Furthermore, in MCF7 cells, despite HER2-normal status and lack of response to single-agent HER2 inhibitors, addition of HER2 inhibitors or dual HER2/EGFR inhibitor to anti-estrogens augments the antiproliferative effect of anti-estrogens, and converts the drug effect from cytostatic to apoptosis-inducing. CONCLUSION: ER-HER co-targeting enhanced the antitumor effects and can bring about effects of targeting HER2 in models of HER2-normal breast cancer.

Salmonella pathogenicity island-2 and anticancer activity in mice.

Salmonella enterica servovar Typhimurium is capable of targeting, colonizing, and eliciting growth suppression of tumors in mice. We examined the effects of mutations on this anticancer phenotype in two Salmonella virulence gene clusters. Salmonella pathogenicity island (SPI)-1 genes promote systemic invasion from the intestine, whereas SPI-2 genes support systemic survival within macrophages and other cells. Disabling SPI-1 (prgH(-)) strongly reduced invasion in vitro, but had no effect on tumor growth suppression in vivo. However, disabling SPI-2 (ssaT(-)) ablated tumor growth suppression. In addition to ssaT(-), mutations in SPI-2 genes sseA, sseB, sseC, sscA, and ssrA also eliminated antitumor activity, whereas mutations in sseF or sseG yielded partial loss of function. Impaired tumor amplification was seen in three SPI-2 mutants tested after intravenous or intratumoral injection. A SPI-2(-) strain was unable to suppress tumor growth in CD18-deficient mice with defective macrophages and neutrophils, suggesting that loss of tumor growth suppression in wild-type mice by SPI-2 mutants was not solely a function of increased susceptibility to immune attack. Thus, SPI-2 is essential for the Salmonella antitumor effects, perhaps by aiding bacterial amplification within tumors, and is the first identified genetic system for this Salmonella phenotype.

Expression of c-Met proto-oncogene in metastatic macrophage x melanoma fusion hybrids: implication of its possible role in MSH-induced motility.

It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed macrophage-specific glycosylation, especially increased GnT-V activity, beta1,6 branch formation in glycoproteins, accompanied by enhanced metastatic potential in vivo and motility in vitro. These hybrids also express upregulated melanocortin-1 receptor (MC1-R) activity and exhibit increased motility after melanocyte-stimulating hormone (MSH) treatment. In this report, we show that MSH-mediated stimulation of motility is mediated through enhanced expression of c-Met proto-oncogene. In metastatic hybrids c-Met expression is induced by MSH, and addition of c-Met neutralizing antibody to cells inhibits MSH-induced motility but not the basal motility of the cells. Furthermore, abrogation of the chemoattractant gradient concentration by addition of hepatocyte growth factor (HGF) recombinant protein, a cognate ligand of c-Met receptor, reduces the MSH-induced effect on motility. A similar result was also obtained by the addition of blocking anti-alphaHGF antibody in the chemoattractant chamber. Again, the metastatic hybrids, but not the nonmetastatic hybrids or parental melanoma cells, showed significant motile response to rHGF chemoattractant, and that motility is further induced when cells were stimulated with MSH/isobutylmethyl xanthine (IBMX). Synergistic stimulation on motility was also observed with those hybrids treated with MSH/IBMX and when rHGF and fibronectin (FN), in combination, were used as chemoattractants. These indicate that MSH/IBMX-induced motility might involve c-Met pathways as well as extracellular matrix (ECM)/integrin pathways in a cooperative fashion. Ets-1, a transcription factor involved in the expression of c-Met, is also found to be induced in metastatic hybrids after exposure to MSH/IBMX. Implication of the result is discussed in light of the role of c-Met and its interacting proteins in the development of metastatic phenotypes and its therapeutic intervention.

Upregulation of alpha and beta integrin subunits in metastatic macrophage-melanoma fusion hybrids.

Fusion of cancer cells with migratory bone-marrow-derived cells such as macrophages can produce cancer cells with increased metastatic potential. To study this, we fused mouse macrophages with weakly metastatic mouse melanoma cells and generated a panel of hybrid clones. About half of these showed increased metastatic potential in mice. These hybrids expressed traits and molecules that were known indicators of tumor progression in melanoma (chemotaxis toward fibronectin, melanogenesis, autophagy, cMet, MCR1, SPARC, cell surface LAMP-1, GnT-V and ß1,6-branched oligosaccharides). Here, we investigated integrin subunit expression in selected hybrids. Integrins, especially those that are substrates for the glycosyltransferase GnT-V and carriers of ß1,6-branched oligosaccharides, play an important role in cell migration. We report increased expression of the integrin subunits α3, α5, α6, αv, ß1, and ß3 in metastatic hybrids compared with parental melanoma cells and a weakly metastatic hybrid. Notably, each of these subunits is also a substrate for GnT-V. Integrin subunit expression was further increased by inducers of cyclic AMP. Expression of these integrin subunits is a characteristic of macrophages and also associated with progression in melanoma and other cancers. In summary, our studies of macrophage-melanoma hybrids show that several α and ß integrin subunits are upregulated in the metastatic lines. This adds further support for the theory that generation of a metastatic phenotype may be initiated through a single event: fusion of migratory bone marrow-derived cells with cancer cells.

Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis.

The causes of metastasis remain elusive despite vast information on cancer cells. We posit that cancer cell fusion with macrophages or other migratory bone marrow-derived cells (BMDCs) provides an explanation. BMDC-tumour hybrids have been detected in numerous animal models and recently in human cancer. Molecular studies indicate that gene expression in such hybrids reflects a metastatic phenotype. Should BMDC-tumour fusion be found to underlie invasion and metastasis in human cancer, new approaches for therapy would surely follow.

Co-targeting insulin-like growth factor I receptor and HER2: dramatic effects of HER2 inhibitors on nonoverexpressing breast cancer.

The insulin-like growth factor I receptor (IGFIR) and HER2 display important signaling interactions in breast cancer. We examined the effect of combinations of antagonists of these receptors using two human breast cancer cell lines: BT474 (HER2+, IGFIR low) and MCF7 (HER2 low, IGFIR high). In BT474 cells, growth was inhibited by HER2 antagonists but not by IGFIR antagonists; however, IGFIR antagonists enhanced the effect of HER2 inhibitors. In MCF7 cells, growth was inhibited by IGFIR antagonists but not by HER2 antagonists; however, HER2 antagonism enhanced the effect of IGFIR inhibitors. Synergistic inhibition of soft agar growth was also observed. Although HER2 and IGFIR antagonists individually only minimally affected cell cycle, their combination gave a small enhancement of their effects. No single receptor-targeting drug was capable of inducing apoptosis, but combining antagonists of both receptors induced a dramatic degree of apoptosis in both cell lines. Induction of apoptosis was most striking in MCF7 cells using a Herceptin/IGFIR antagonist combination despite these cells being HER2 nonoverexpressing. Toward understanding the mechanism of these effects, we detected coassociation IGFIR and HER2 in both cell lines. Specific inhibitors of one of these receptors could cross-inhibit the activity of the other. Targeting both receptors gave the maximal inhibition of their downstream extracellular signal-regulated kinase 1/2 and AKT signaling pathways. Hence, such drug combinations may be clinically useful and may be beneficial even in tumors in which single drugs are inactive, as exemplified by the effect of the HER2/IGFIR inhibitor combination in HER2 nonoverexpressing MCF7 cells.

Evidence that the small GTPase Rab8 is involved in melanosome traffic and dendrite extension in B16 melanoma cells.

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion.