Epithelial requirement for in vitro proliferation and xenograft growth and metastasis of MDA-MB-468 human breast cancer cells: oncogenic rather than tumor-suppressive role of E-cadherin.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice. METHODS: EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student's t tests were performed to determine significance (p < 0.05). RESULTS: EMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of these cells were globally vimentin-positive rather than exhibiting regional EMT, and they expressed higher SNA1 than their in vitro counterparts. E-cadherin suppression caused a trend toward reduced lung metastasis, whereas E-cadherin overexpression resulted in the reverse trend, consistent with the increased proliferation rate and predominantly epithelial phenotype of MDA-MB-468 cells outside the primary xenograft. This was also originally observed in WT xenografts. Furthermore, we found that patients with breast cancer that expressed E-cadherin were more likely to have metastases. CONCLUSIONS: E-cadherin expression promotes growth of primary breast tumors and conceivably the formation of metastases, supporting a role for MET in metastasis. E-cadherin needs to be reevaluated as a tumor suppressor.

Effects of Tamoxifen and oestrogen on histology and radiographic density in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers.

Mammographic density (MD) is a strong risk factor for breast cancer. It is altered by exogenous endocrine treatments, including hormone replacement therapy and Tamoxifen. Such agents also modify breast cancer (BC) risk. However, the biomolecular basis of how systemic endocrine therapy modifies MD and MD-associated BC risk is poorly understood. This study aims to determine whether our xenograft biochamber model can be used to study the effectiveness of therapies aimed at modulating MD, by examine the effects of Tamoxifen and oestrogen on histologic and radiographic changes in high and low MD tissues maintained within the biochamber model. High and low MD human tissues were precisely sampled under radiographic guidance from prophylactic mastectomy fresh specimens of high-risk women, then inserted into separate vascularized murine biochambers. The murine hosts were concurrently implanted with Tamoxifen, oestrogen or placebo pellets, and the high and low MD biochamber tissues maintained in the murine host environment for 3 months, before the high and low MD biochamber tissues were harvested for histologic and radiographic analyses. The radiographic density of high MD tissue maintained in murine biochambers was decreased in Tamoxifen-treated mice compared to oestrogen-treated mice (p = 0.02). Tamoxifen treatment of high MD tissue in SCID mice led to a decrease in stromal (p = 0.009), and an increase in adipose (p = 0.023) percent areas, compared to placebo-treated mice. No histologic or radiographic differences were observed in low MD biochamber tissue with any treatment. High MD biochamber tissues maintained in mice implanted with Tamoxifen, oestrogen or placebo pellets had dynamic and measurable histologic compositional and radiographic changes. This further validates the dynamic nature of the MD xenograft model, and suggests the biochamber model may be useful for assessing the underlying molecular pathways of Tamoxifen-reduced MD, and in testing of other pharmacologic interventions in a preclinical model of high MD.

Dynamic changes in high and low mammographic density human breast tissues maintained in murine tissue engineering chambers during various murine peripartum states and over time.

Mammographic density (MD) is a strong heritable risk factor for breast cancer, and may decrease with increasing parity. However, the biomolecular basis for MD-associated breast cancer remains unclear, and systemic hormonal effects on MD-associated risk is poorly understood. This study assessed the effect of murine peripartum states on high and low MD tissue maintained in a xenograft model of human MD. Method High and low MD human breast tissues were precisely sampled under radiographic guidance from prophylactic mastectomy specimens of women. The high and low MD tissues were maintained in separate vascularised biochambers in nulliparous or pregnant SCID mice for 4 weeks, or mice undergoing postpartum involution or lactation for three additional weeks. High and low MD biochamber material was harvested for histologic and radiographic comparisons during various murine peripartum states. High and low MD biochamber tissues in nulliparous mice were harvested at different timepoints for histologic and radiographic comparisons. Results High MD biochamber tissues had decreased stromal (p = 0.0027), increased adipose (p = 0.0003) and a trend to increased glandular tissue areas (p = 0.076) after murine postpartum involution. Stromal areas decreased (p = 0.042), while glandular (p = 0.001) and adipose areas (p = 0.009) increased in high MD biochamber tissues during lactation. A difference in radiographic density was observed in high (p = 0.0021) or low MD biochamber tissues (p = 0.004) between nulliparous, pregnant and involution groups. No differences in tissue composition were observed in high or low MD biochamber tissues maintained for different durations, although radiographic density increased over time. Conclusion High MD biochamber tissues had measurable histologic changes after postpartum involution or lactation. Alterations in radiographic density occurred in biochamber tissues between different peripartum states and over time. These findings demonstrate the dynamic nature of the human MD xenograft model, providing a platform for studying the biomolecular basis of MD-associated cancer risk.

High and low mammographic density human breast tissues maintain histological differential in murine tissue engineering chambers.

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal (p = 0.0002) and lower adipose (p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area (p = 0.4) or count (p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer.

Epithelial mesenchymal transition traits in human breast cancer cell lines.

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

Correlation of tumor- and stromal-derived MT1-MMP expression with progression of human ovarian tumors in SCID mice.

OBJECTIVE: Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. METHODS: Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. RESULTS: Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. CONCLUSIONS: These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

Biochemical Methods for the Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

Viruses that are less sensitive to the influenza neuraminidase (NA)-specific inhibitor 4-guanidino-Neu5Ac2en (zanamavir) (1) can be isolated after several passages in MDCK cells in the presence of the inhibitor. Although there are three reports of a mutation in the NA gene at the same conserved site, glu119 (2-4), most of the variants have mutations in the hemagglutinin (HA) gene (5). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. In this chapter we describe noncell culture-based methods for characterization of both HA and NA variants.

Virological Methods for the Generation and Characterization of Influenza Viruses with Reduced Sensitivity to 4-Guanidino-Neu5Ac2en.

The compound 4-guanidino-Neu5Ac2en (zanamivir) has been described as a selective inhibitor of the influenza virus neuraminidase (NA) (1). Viruses that are less sensitive to this inhibitor can be isolated after several passages in MDCK cells in the presence of the inhibitor. Variants isolated so far have had mutations predominantly in the hemagglutinin (HA) gene (2). Many of these mutations appear to lower the affinity of the HA for the cellular receptor, so that there is less requirement for significant NA activity for the newly synthesized progeny virus to elute. There are three reports of a mutation in the NA gene, all at the same conserved site, glu 119 (3-5). In this chapter, the authors describe methods for the isolation of the mutants, and for their characterization in cell culture based assays.

The interaction of neuraminidase and hemagglutinin mutations in influenza virus in resistance to 4-guanidino-Neu5Ac2en.

We have previously described a 4-guanidino-Neu5Ac2en (zanamivir)-resistant neuraminidase (NA) variant G70C4-G, with an active site mutation Glu 119 to Gly. This variant has been found to also harbor a hemagglutinin (HA) mutation in the receptor binding site, Ser 186 to Phe. Examination of early passages of the G70C4-G virus revealed that this HA mutation had arisen by the first passage. From a subsequent passage two transient variants were isolated which had each acquired a different second HA mutation, Ser 165 to Asn and Lys 222 to Thr. Both were highly drug resistant and drug dependent and their ability to adsorb to and penetrate cells was decreased. Comparison of drug sensitivities between the variant, with the additional HA mutation at Ser 165, and viruses with either mutation alone revealed that these two HA mutations acted synergistically to increase resistance. To determine the contribution to resistance of each of the NA and HA mutations in G70C4-G, the NA mutation was separated from the HA mutation by reassorting. The NA mutation and the HA mutation each conferred low-level resistance to zanamivir, while the two mutations interacted synergistically in the double mutant to give higher resistance in vitro. Infectivity was not adversely affected in the double mutant and while there was a small decrease in sensitivity to zanamivir in the mouse model, there was no detectable resistance to zanamivir in the ferret model.

Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase.

BACKGROUND: Inhibitors of the influenza virus neuraminidase have been shown to be effective antiviral agents in humans. Several studies have reported the selection of novel influenza strains when the virus is cultured with neuraminidase inhibitors in vitro. These resistant viruses have mutations either in the neuraminidase or in the viral haemagglutinin. Inhibitors in which the glycerol sidechain at position 6 of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) has been replaced by carboxamide-linked hydrophobic substituents have recently been reported and shown to select neuraminidase variants. This study seeks to clarify the structural and functional consequences of replacing the glycerol sidechain of the inhibitor with other chemical constituents. RESULTS: The neuraminidase variant Arg292-->Lys is modified in one of three arginine residues that encircle the carboxylate group of the substrate. The structure of this variant in complex with the carboxamide inhibitor used for its selection, and with other Neu5Ac2en analogues, is reported here at high resolution. The structural consequences of the mutation correlate with altered inhibitory activity of the compounds compared with wild-type neuraminidase. CONCLUSIONS: The Arg292-->Lys variant of influenza neuraminidase affects the binding of substrate by modification of the interaction with the substrate carboxylate. This may be one of the structural correlates of the reduced enzyme activity of the variant. Inhibitors that have replacements for the glycerol at position 6 are further affected in the Arg292-->Lys variant because of structural changes in the binding site that apparently raise the energy barrier for the conformational change in the enzyme required to accommodate such inhibitors. These results provide evidence that a general strategy for drug design when the target has a high mutation frequency is to design the inhibitor to be as closely related as possible to the natural ligands of the target.

Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors.

The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.

Structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases.

The x-ray structure of a complex of sialic acid (Neu5Ac) with neuraminidase N9 subtype from A/tern/Australia/G70C/75 influenza virus at 4 degrees C has revealed the location of a second Neu5Ac binding site on the surface of the enzyme. At 18 degrees C, only the enzyme active site contains bound Neu5Ac. Neu5Ac binds in the second site in the chair conformation in a similar way to which it binds to hemagglutinin. The residues that interact with Neu5Ac at this second site are mostly conserved in avian strains, but not in human and swine strains, indicating that it has some as-yet-unknown biological function in birds.

Mutation in the influenza virus neuraminidase gene resulting in decreased sensitivity to the neuraminidase inhibitor 4-guanidino-Neu5Ac2en leads to instability of the enzyme.

We previously isolated a variant of the influenza virus NWS/G70C, with a decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en in vitro, which has a mutation in one of the conserved residues of the neuraminidase Glu 119 to Gly. Despite the mutation, purified neuraminidase demonstrated the same specific activity as the parent neuraminidase. In contrast, characterization of a similar mutant by another group revealed a low specific activity of the enzyme. We confirm here that the specific activity of our variant is the same as that of the parent, but report that this mutation makes the enzyme inherently unstable, at high and low temperatures, either on the virion or as purified neuraminidase. Thus, for a valid determination of specific activity the concentration of native NA needs to be determined at the time of enzyme assay. Structurally, the instability may be partially explained by the introduction of a side chain (Gly), which carries a greater entropy penalty in condensation of the structure from the unfolded to the folded state and this, together with the loss of stabilizing interaction between Glu 119 and its neighbors in the active site, is not compensated for by the water molecule occupying the position of the carboxylate group (6).

Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en.

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en.

A variant of the influenza virus NWS/G70C has been generated which has decreased sensitivity in vitro to the neuraminidase-specific inhibitor, 4-guanidino-Neu5Ac2en. The virus is 1000-fold less sensitive to the 4-guanidino-Neu5Ac2en in a plaque assay, but only 10-fold less sensitive to 4-amino-Neu5Ac2en. In an enzyme inhibition assay 250-fold more drug was needed to achieve inhibition comparable to that observed with the parent virus. In contrast to the plaque assay, the virus was fully sensitive to 4-amino-Neu5Ac2en in the enzyme inhibition assay. Kinetic analysis of 4-guanidino-Neu5Ac2en binding demonstrated that the variant no longer exhibited the slow binding characteristic seen with the parent and other influenza viruses and inhibition by Neu5Ac2en was also decreased. However, binding to 4-amino-Neu5Ac2en remained the same as the parent. Sequence analysis of this virus revealed a mutation at a previously conserved site in the enzyme active site of the neuraminidase, Glu 119 to Gly. Crystallographic analysis of the mutant neuraminidase with and without bound inhibitor confirmed this mutation and suggested that the reduced affinity for the 4-guanidino-Neu5Ac2en derives partly from the loss of a stabilizing interaction between the guanidino moiety and the carboxylate at residue 119, and partly from alterations to the solvent structure of the active site.