J. Louise Jones

Carbonic Anhydrase IX Expression, a Novel Surrogate Marker of Tumor Hypoxia, Is Associated With a Poor Prognosis in Non–Small-Cell Lung Cancer

PURPOSE To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). METHODS Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. RESULTS CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P =.005), cytoplasmic (c) (P =.018), and stromal (P <.001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 micro m, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P <.001). Hypoxic exposure of A549 cells for 16 hours enhanced CA IX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P =.035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P =.004), stage (P =.001), platelet count (P =.011), sex (P =.027), and TN (P =.035) were independent poor prognostic factors. CONCLUSION These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype.

Expression of MMP‐2 and MMP‐9, their inhibitors, and the activator MT1‐MMP in primary breast carcinomas

Enhanced expression of the type IV collagenases MMP‐2 and MMP‐9, or lack of their inhibitors TIMP‐1 and TIMP‐2, has been associated with tumour invasion and metastatic potential in several experimental models. Regulation of enzyme activity is clearly a key step in tumour invasion, and recently a potent activator of MMP‐2, the membrane‐associated MT1‐MMP, has been described and characterized. Using an immunohistochemical approach, this study has examined the expression and distribution of the type IV collagenases, their inhibitors, and the activator MT1‐MMP, in a series of 79 infiltrating ductal carcinomas (IDCs), 8 tubular carcinomas, and 27 infiltrating lobular carcinomas (ILCs). MMP‐2 and MT1‐MMP were expressed in more than 90 per cent of all carcinomas, with predominantly stromal and tumour cell cytoplasmic staining. However, reactivity localized on tumour cell membranes was recorded for MMP‐2 in 34 per cent of cases with a monoclonal antibody and 55 per cent of cases with a polyclonal antibody, and for MT1‐MMP in 68 per cent of tumours. In each case, this pattern of staining was significantly associated with the presence of lymph node metastasis ( p=0·001, p=0·008, and p=0·1, respectively). Both tumour cell and stromal staining was observed for TIMP‐2, but there was no correlation with metastatic status. The 92 kD gelatinase MMP‐9 was expressed by 68 per cent of carcinomas, either in the stromal compartment or by tumour cells. There was a highly significant correlation between the expression pattern of MMP‐9 and tumour type, with ILCs displaying greater frequency and more homogeneous cytoplasmic staining than IDCs ( p=0·0004). Staining for TIMP‐1 was seen in the stroma and also in relation to small blood vessels, with more than 90 per cent of tumours showing this staining pattern using a polyclonal antibody. This study indicates distinct patterns of expression for different MMPs and demonstrates the potential importance of the MMP‐2/MT1‐MMP system in breast tumour progression. The association of MMP‐9 with the infiltrating lobular phenotype may reveal novel mechanisms of control for this metalloproteinase. Copyright

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

Dysregulated expression of adamalysin-thrombospondin genes in human breast carcinoma.

The adamalysin-thrombospondin (ADAMTS) proteinases are a relatively newly described branch of the metzincin family that contain metalloproteinase, disintegrin, and thrombospondin motifs. They have been implicated in various cellular events, including cleavage of proteoglycans, extracellular matrix degradation, inhibition of angiogenesis, gonadal development, and organogenesis. However, in many cases, their normal physiological roles and their potential for dysregulation in malignancy remain to be established. The expression profile of ADAMTS1–20 in human breast carcinoma was undertaken by real-time PCR using RNA isolated from malignant tumors, nonneoplastic mammary tissue, and breast cancer cell lines to identify altered regulation that may have potential pathogenetic and prognostic significance. Our studies show that seven of the ADAMTS genes (ADAMTS1, 3, 5, 8, 9, 10, and 18) are consistently down-regulated in breast carcinomas with respect to nonneoplastic mammary tissue, irrespective of the heterogeneity of the samples and the tumor type or grade (Mann-Whitney U test, P < 0.0001 for each gene). Conversely, ADAMTS4, 6, 14, and 20 are consistently up-regulated in breast carcinomas (P = 0.005, P < 0.0001, P = 0.003, and P = 0.001, respectively). ADAMTS2, 7, 12, 13, 15, 16, 17, and 19 show no significant difference between the sample types. ADAMTS1, 2, 7, 8, 10, and 12 are expressed predominantly in stromal fibroblasts. ADAMTS3, 4, 5, 6, 9, and 13–20 inclusive are expressed predominantly in myoepithelial cells; all appear to be relatively poorly expressed in luminal epithelial cells. ADAMTS15 has emerged as being an independent predictor of survival, with RNA expression levels significantly lower (P = 0.007) in grade 3 breast carcinoma compared with grade 1 and 2 breast carcinoma.

Hypoxia-inducible factor-1α in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathways

Hypoxia‐inducible factor (HIF)‐1α is the regulatory subunit of HIF‐1 that is stabilized under hypoxic conditions. Under different circumstances, HIF‐1α may promote both tumorigenesis and apoptosis. There is conflicting data on the importance of HIF‐1α as a prognostic factor. This study evaluated HIF‐1α expression in 172 consecutive patients with stage I–IIIA non small cell lung cancer (NSCLC) using standard immunohistochemical techniques. The extent of HIF‐1α nuclear immunostaining was determined using light microscopy and the results were analyzed using the median (5%) as a low cut‐point and 60% as a high positive cut‐point. Using the low cut‐point, positive associations were found with epidermal growth factor receptor (EGFR; p = 0.01), matrix metalloproteinase (MMP)‐9 (p = 0.003), membranous (p < 0.001) and perinuclear (p = 0.004) carbonic anhydrase (CA) IX, p53 (p = 0.008), T‐stage (p = 0.042), tumor necrosis (TN; p < 0.001) and squamous histology (p < 0.001). No significant association was found with Bcl‐2 or either N‐ or overall TMN stage or prognosis. When the high positive cut‐point was used, HIF‐1α was associated with a poor prognosis (p = 0.034). In conclusion, the associations with EGFR, MMP‐9, p53 and CA IX suggest that these factors may either regulate or be regulated by HIF‐1α. The association with TN and squamous‐type histology, which is relatively more necrotic than other NSCLC types, reflects the role of hypoxia in the regulation of HIF‐1α. The prognostic data may reflect a change in the behavior of HIF‐1α in increasingly hypoxic environments.

Rigidity sensing and adaptation through regulation of integrin types.

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5β1 integrins (constitutively expressed) or αvβ6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow, and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin.

Breast cell invasive potential relates to the myoepithelial phenotype

On the basis of marker profile, the majority of breast carcinomas are thought to be derived from luminal epithelial cells; however, a subgroup of tumours with more mesenchymal characteristics are associated with a worse prognosis. The hypothesis of our study is that some breast carcinomas exhibit myoepithelial rather than pure mesenchymal differentiation and that acquisition of myoepithelial characteristics confers an aggressive phenotype. Pure luminal epithelial cells and fibroblasts are readily distinguished by many markers but distinguishing between myoepithelial and fibroblast cell lineages is more problematic. The markers found to be most discriminating in our study were CK14, α6β4 integrin and the myoepithelial‐associated desmosomal cadherin DSg3. These markers were applied to a series of breast cell lines and purified normal breast cell populations and the expression profile related to in vitro invasive behaviour. This demonstrated that expression of one or more myoepithelial markers by tumour cells (MDA MB 231, MDA MB 468, MDA MB 436) was associated with a high invasive capacity compared with cells with a pure luminal phenotype (MCF‐7, T47D, ZR75). To address why myoepithelial characteristics are associated with higher invasion, the in vitro behaviour of normal myoepithelial cells and two other nontumourigenic breast cell lines (MCF‐10A, HBL100) was also analysed. Primary myoepithelial cells from normal human breast exhibit a high invasive capacity when grown at low density, suggesting that invasive capacity is part of the myoepithelial phenotype. In keeping with this, both nontumourigenic cell lines exhibited features of the myoepithelial phenotype and a high invasive capacity. These results suggest that tumours that exhibit a myoepithelial phenotype may be clinically more aggressive because a high invasive capacity is intrinsic to the myoepithelial phenotype.

Oestrogen receptors alpha and beta differ in normal human breast and breast carcinomas

The identification of a second oestrogen receptor, oestrogen receptor (ER) β, has led to a need to assess the relative importance of the classical ERα and ERβ in human breast and breast carcinomas. ERα and ERβ mRNA was assessed in 61 carcinomas, 8 benign breast lesions, and 30 samples of normal breast using reverse transcriptase (RT)‐nested polymerase chain reaction (PCR). Immunohistochemical analysis of ERα and ERβ was performed in 62 carcinomas, the 38 non‐malignant breast tissues, and 32 normal breast samples with menstrual cycle data. ERα mRNA was detected in 92% of breast cancers, with ERβ mRNA (wild‐type and/or variant form) in 85%; 72% had ERα protein, 62% progesterone receptor (PgR), and 32% ERβ. ERα protein had a strong correlation with grade; ERβ did not, although it was present in three of four grade I carcinomas and in special types. There was no correlation between the presence of ERα and ERβ protein. In non‐malignant breast, similar expression of ERα and β was observed, apart from expression of ERβ in stromal cells and myoepithelium, the latter being confirmed by RT‐PCR and western blotting. There were differences in ERα in relation to the menstrual cycle but not PgR or ERβ. The findings indicate a need to understand the role and regulation of ERβ in normal breast and the reason for its down‐regulation in mammary carcinogenesis. Copyright

Tumour necrosis is an independent prognostic marker in non-small cell lung cancer: correlation with biological variables.

BACKGROUND Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. METHODS In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. RESULTS Reproducibility was attained in 36/40 cases, Kappa score = 0.8 P < 0.001. TN correlated with T-stage (P = 0.001), platelet count (P = 0.004) and p53 expression (P = 0.031). Near significant associations of TN with N-stage (P = 0.063) and MMP-9 expression (P = 0.058) were seen. No association was found with angiogenesis (P = 0.98). On univariate (P = 0.0016) and multivariate analysis (P = 0.023) TN was prognostic. CONCLUSION These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections.

Tumour-associated tenascin-C isoforms promote breast cancer cell invasion and growth by matrix metalloproteinase-dependent and independent mechanisms

IntroductionThe stromal microenvironment has a profound influence on tumour cell behaviour. In tumours, the extracellular matrix (ECM) composition differs from normal tissue and allows novel interactions to influence tumour cell function. The ECM protein tenascin-C (TNC) is frequently up-regulated in breast cancer and we have previously identified two novel isoforms – one containing exon 16 (TNC-16) and one containing exons 14 plus 16 (TNC-14/16).MethodsThe present study has analysed the functional significance of this altered TNC isoform profile in breast cancer. TNC-16 and TNC-14/16 splice variants were generated using PCR-ligation and over-expressed in breast cancer cells (MCF-7, T47D, MDA-MD-231, MDA-MB-468, GI101) and human fibroblasts. The effects of these variants on tumour cell invasion and proliferation were measured and compared with the effects of the large (TNC-L) and fully spliced small (TNC-S) isoforms.ResultsTNC-16 and TNC-14/16 significantly enhanced tumour cell proliferation (P < 0.05) and invasion, both directly (P < 0.01) and as a response to transfected fibroblast expression (P < 0.05) with this effect being dependent on tumour cell interaction with TNC, because TNC-blocking antibodies abrogated these responses. An analysis of 19 matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases 1 to 4 (TIMP 1 to 4) revealed that TNC up-regulated expression of MMP-13 and TIMP-3 two to four fold relative to vector, and invasion was reduced in the presence of MMP inhibitor GM6001. However, this effect was not isoform-specific but was elicited equally by all TNC isoforms.ConclusionsThese results demonstrate a dual requirement for TNC and MMP in enhancing breast cancer cell invasion, and identify a significant role for the tumour-associated TNC-16 and TNC-14/16 in promoting tumour invasion, although these isoform-specific effects appear to be mediated through MMP-independent mechanisms.