Angiogenesis and non-small cell lung cancer

Lung cancer is the commonest cause of cancer death in the western world. Recent evidence suggests that angiogenesis is related to poor prognosis in many solid tumours including non-small cell lung cancer. Angiogenesis is controlled by a complex interaction between growth and apoptotic factors, proteases and adhesion molecules. The angiogenic process may prove a target for novel therapies such as matrix metalloproteinase inhibitors, growth factor antisense RNA, growth factor receptor antagonists and naturally occurring antiangiogenic peptides. These agents may be used alone or in combination with traditional chemotherapy, radiotherapy and surgery. (C) 2000 Elsevier Science Ireland Ltd.

Prognostic significance of platelet and microvessel counts in operable non-small cell lung cancer

Background: Microvessel density, an indirect measure of angiogenesis, has been shown to be an independent prognostic marker in many solid tumours including non-small cell lung cancer (NSCLC). Platelets transport and release angiogenic growth factors. Platelets are increasingly likely to adhere to tumour microvessels due to raised expression of platelet-binding proteins and stasis in blood-flow. Increased vascular permeability in tumour microvessels facilitates platelet extravasation into the extracellular matrix. Adherence and extravasation both lead to platelet activation and release of growth factors capable of instigating the angiogenic process. Methods: A total of 181 patients were identified who underwent resection of stage I-IIIa NSCLC with a post-operative survival >60 days. Patients were followed-up for a minimum of 24 months. Sections from the tumour periphery were stained for the endothelial marker CD34 (Novocastra NCL-END) using standard ABC immunohistochemistry. Chalkley counting was used to assess microvessel density. Results: A pre-operative platelet count greater than the median and above the normal range (>400) was associated with a poor outcome (P = 0.01 and P = 0.04, respectively). Tumours with an above median and high Chalkley count (upper tertile) had a worse prognosis (P = 0.007 and P = 0.0006, respectively). There was no association between platelet count and Chalkley count. Conclusions: Platelet and microvessel counts are both potential prognostic markers for NSCLC. The role of platelets in the angiogenic process needs to be further investigated. (C) 2000 Elsevier Science Ireland Ltd.

Phase III trial of gemcitabine and carboplatin versus mitomycin, ifosfamide, and cisplatin or mitomycin, vinblastine, and cisplatin in patients with advanced nonsmall cell lung carcinoma

BACKGROUND. The authors compared gemcitabine and carboplatin (GC) with mitomycin, ifosfamide, and cisplatin (MIC) or mitomycin, vinblastine, and cisplatin (MVP) in patients with advanced nonsmall cell lung carcinoma (NSCLC). The primary objective was survival. Secondary objectives were time to disease progression, response rates, evaluation of toxicity, disease-related symptoms, World Health Organization performance status (PS), and quality of life (QoL). METHODS. Three hundred seventy-two chemotherapy-naïve patients with International Staging System Stage III/IV NSCLC who were ineligible for curative radiotherapy or surgery were randomized to receive either 4 cycles of gemcitabine (1000 mg/m2 on Days 1, 8, and 15) plus carboplatin (area under the serum concentration-time curve, 5; given on Day 1) every 4 weeks (the GC arm) or MIC/MVP every 3 weeks (the MIC/MVP arm). RESULTS. There was no significant difference in median survival (248 days in the MIC/MVP arm vs. 236 days in the GC arm) or time to progression (225 days in the MIC/MVP arm vs. 218 days in the GC arm) between the 2 treatment arms. The 2-year survival rate was 11.8% in the MIC/MVP arm and 6.9% in the GC arm. The 1-year survival rate was 32.5% in the MIC/MVP arm and 33.2% in the GC arm. In the MIC/MVP arm, 33% of patients responded (4 complete responses [CRs] and 57 partial responses [PRs]) whereas in the GC arm, 30% of patients responded (3 CRs and 54 PRs). Nonhematologic toxicity was comparable for patients with Grade 3-4 symptoms, except there was more alopecia among patients in the MIC/MVP arm. GC appeared to produce more hematologic toxicity and necessitated more transfusions. There was no difference in performance status, disease-related symptoms, of QoL between patients in the two treatment arms. Fewer inpatient stays for complications were required with GC. CONCLUSIONS. The results of the current study failed to demonstrate any difference in efficacy between the newer regimen of GC and the older regimens of MIC and MVP. © 2003 American Cancer Society.

Platelet-derived endothelial cell growth factor expression and angiogenesis in cervical intraepithelial neoplasia and squamous cell carcinoma of the cervix

Growth and metastatic spread of invasive carcinoma depends on angiogenesis, the formation of new blood vessels. Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic growth factor for a number of solid tumors, including lung, bladder, colorectal, and renal cell cancer. Cervical intraepithelial neoplasia (CIN) is the precursor to squamous cell cervical carcinoma (SCC). Mean vessel density (MVD) increases from normal cervical tissue, through low- and high-grade CIN to SCC. We evaluated PD-ECGF immunoreactivity and correlated its expression with MVD in normal, premalignant, and malignant cervical tissue. PD-ECGF expression was assessed visually within the epithelial tissues and scored on the extent and intensity of staining. MVD was calculated by counting the number of vessels positive for von Willebrand factor per unit area subtending normal or CIN epithelium or within tumor hotspots for SCC. Cytoplasmic and/or nuclear PD-ECGF immunoreactivity was seen in normal epithelium. PD-ECGF expression significantly increased with histologic grade from normal, through low- and high-grade CIN, to SCC (P < .02). A progressive significant increase in the microvessel density was also seen, ranging from a mean of 28 vessels for normal tissue to 57 for SCC (P < .0005). No correlation was found between PD-ECGF expression and MVD (P = .45). We conclude that PD-ECGF expression and MVD increase as the cervix transforms from a normal to a malignant phenotype. PD-ECGF is thymidine phosphorylase, a key enzyme in the activation of fluoropyrimidines, including 5-fluorouracil. Evaluation of PD-ECGF thymidine phosphorylase expression may be important in designing future chemotherapeutic trials in cervical cancer. Copyright (C) 2000 by W.B. Saunders Company.

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5- monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of I mM for six or more hours, significantly enhances the efficacy of 5-FU. Patients and methods: 5-FU/FA was given as follows: days 1 and 2 - FA 250 mg/m 2 (max. 350 mg) over two hours followed by 5-FU 400 mg/m 2 by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m 2 infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours. Results: Thirty patients were entered into the study. Median survival was nine months (range 1-51 + months). There were eight partial responses (28%, 95% CI: 13%-47%). The median duration of response was 6.5 (range 4-9 months). Grade 3-4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another. Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.

Phase II trial of Oxaliplatin and 5-Fluorouracil/Leucovorin combination in epithelial ovarian carcinoma relapsing within 2 years of platinum-based therapy

Objective To evaluate the efficacy and toxicity of Oxaliplatin and 5-Fluorouracil (5-FU)/Leucovorin (LV) combination in ovarian cancer relapsing within 2 years of prior platinum-based chemotherapy in a phase II trial. Methods Eligible patients had at least one prior platinum-based chemotherapy regimen, elevated CA-125 ≥ 60 IU/l, radiological evidence of disease progression and adequate hepatic, renal and bone marrow function. Patients with raised CA-125 levels alone as marker of disease relapse were not eligible. Oxaliplatin (85 mg/m 2) was given on day 1, and 5-Fluorouracil (370 mg/m 2) and Leucovorin (30 mg) was given on days 1 and 8 of a 14-day cycle. Results Twenty-seven patients were enrolled. The median age was 57 years (range 42-74 years). The median platinum-free interval (PFI) was 5 months (range 0-17 months) with only 30% of patients being platinum sensitive (PFI > 6 months). Six patients (22%) had two prior regimens of chemotherapy. A total of 191 cycles were administered (median 7; range 2-12). All patients were evaluable for toxicity. The following grade 3/4 toxicities were noted: anemia 4%; neutropenia 15%; thrombocytopenia 11%; neurotoxicity 8%; lethargy 4%; diarrhea 4%; hypokalemia 11%; hypomagnesemia 11%. Among 27 enrolled patients, 20 patients were evaluable for response by WHO criteria and 25 patients were evaluable by Rustin's CA-125 criteria. The overall response rate (RR) by WHO criteria was 30% (95% CI: 15- 52) [three complete responses (CRs) and three partial responses (PRs)]. The CA-125 response rate was 56% (95% CI: 37-73). Significantly, a 25% (95% CI: 9-53) radiological and a 50% (95% CI: 28-72) CA-125 response rate were noted in platinum resistant patients (PFI < 6 months). The median response duration was 4 months (range 3-12) and the median overall survival was 10 months. Conclusion Oxaliplatin and 5-Fluorouracil/ Leucovorin combination has a good safety profile and is active in platinum-pretreated advanced epithelial ovarian cancer. © 2004 Elsevier Inc. All rights reserved.

Interactions between hypoxia and epidermal growth factor receptor in non-small-cell-lung cancer

Tumor hypoxia has been recognized to confer resistance to anticancer therapy since the early 20th century. More recently, its fundamental role in tumorigenesis has been established. Hypoxia-inducible factor (HIF)-1 has been identified as an important transcription factor that mediates the cellular response to hypoxia, promoting both cellular survival and apoptosis under different conditions. Increased tumor cell expression of this transcription factor promotes tumor growth In vivo and is associated with a worse prognosis in patients with non-small-cell lung cancer (NSCLC) undergoing tumor resection. The epidermal growth factor receptor (EGFR) promotes tumor cell proliferation and anglogenesis and inhibits apoptosis. Epidermal growth factor receptor expression increases in a stepwise manner during tumorigenesis and is overexpressed in > 50% of NSCLC tumors. This review discusses the reciprocal relationship between tumor cell hypoxia and EGFR. Recent studies suggest that hypoxia induces expression of EGFR and its ligands. In return, EGFR might enhance the cellular response to hypoxia by increasing expression of HIF-1α, and so act as a survival factor for hypoxic cancer cells. Immunohistochemical studies on a series of resected NSCLC tumors add weight to this contention by demonstrating a close association between expression of EGFR, HIF-1α, and:1 of HIF-1's target proteins, carbonic anhydrase IX. In this article we discuss emerging treatment strategies for NSCLC that target HIF-1, HIF-1 transcriptional targets, and EGFR.

Strategies and challenges for systematically mapping biologically significant molecular pathways regulating carcinoma epithelial-mesenchymal transition

Enormous progress has been made towards understanding the role of specific factors in the process of epithelial-mesenchymal transition (EMT); however, the complex underlying pathways and the transient nature of the transition continues to present significant challenges. Targeting tumour cell plasticity underpinning EMT is an attractive strategy to combat metastasis. Global gene expression profiling and high-content analyses are among the strategies employed to identify novel EMT regulators. In this review, we highlight several approaches to systematically interrogate key pathways involved in EMT, with particular emphasis on the features of multiparametric, high-content imaging screening strategies that lend themselves to the systematic discovery of highly significant modulators of tumour cell plasticity.

The epidemiology of basal cell carcinoma

Basal cell carcinoma (BCC) is a skin cancer of particular importance to the Australian community. Its rate of occurrence is highest in Queensland, where 1% to 2% of people are newly affected annually. This is an order of magnitude higher than corresponding incidence estimates in European and North American populations. Individuals with a sun-sensitive complexion are particularly susceptible because sun exposure is the single most important causative agent, as shown by the anatomic distribution of BCC which is in general consistent with the levels of sun exposure across body sites. A distinguishing feature of BCC is the occurrence of multiple primary tumours within individuals, synchronously or over time, and their diagnosis and treatment costs contribute substantially to the major public health burden caused by BCC. A primary knowledge gap about BCC pathogenesis however was an understanding of the true frequency of multiple BCC occurrences and their body distribution, and why a proportion of people do develop more than one BCC in their life. This research project sought to address this gap under an overarching research aim to better understand the detailed epidemiology of BCC with the ultimate goal of reducing the burden of this skin cancer through prevention. The particular aim was to document prospectively the rate of BCC occurrence and its associations with constitutional and environmental (solar) factors, all the while paying special attention to persons affected by more than one BCC. The study built on previous findings and recent developments in the field but set out to confirm and extend these and propose more adequate theories about the complex epidemiology of this cancer. Addressing these goals required a new approach to researching basal cell carcinoma, due to the need to account for the phenomenon of multiple incident BCCs per person. This was enabled by a 20 year community-based study of skin cancer in Australians that provided the methodological foundation for this thesis. Study participants were originally randomly selected in 1986 from the electoral register of all adult residents of the subtropical township of Nambour in Queensland, Australia. On various occasions during the study, participants were fully examined by dermatologists who documented cumulative photodamage as well as skin cancers. Participants completed standard questionnaires about skin cancer-related factors, and consented to have any diagnosed skin cancers notified to the investigators by regional pathology laboratories in Queensland. These methods allowed 100% ascertainment of histologically confirmed BCCs in this study population. 1339 participants had complete follow-up to the end of 2007. Statistical analyses in this thesis were carried out using SAS and SUDAAN statistical software packages. Modelling methods, including multivariate logistic regressions, allowed for repeated measures in terms of multiple BCCs per person. This innovative approach gave new findings on two levels, presented in five chapters as scientific papers: 1. Incidence of basal cell carcinoma multiplicity and detailed anatomic distribution: longitudinal study of an Australian population The incidence of people affected multiple times by BCC was 705 per 100,000 person years compared to an incidence rate of people singly affected of 935 per 100,000 person years. Among multiply and singly affected persons alike, site-specific BCC incidence rates were far highest on facial subsites, followed by upper limbs, trunk, and then lower limbs 2. Melanocytic nevi and basal cell carcinoma: is there an association? BCC risk was significantly increased in those with forearm nevi (Odds Ratios (OR) 1.43, 95% Confidence Intervals (CI) 1.09-1.89) compared to people without forearm nevi, especially among those who spent their time mainly outdoors (OR 1.6, 95%CI 1.1-2.3) compared to those who spent their time mainly indoors. Nevi on the back were not associated with BCC. 3. Clinical signs of photodamage are associated with basal cell carcinoma multiplicity and site: a 16-year longitudinal study Over a 16-year follow-up period, 58% of people affected by BCC developed more than one BCC. Among these people 60% developed BCCs across different anatomic sites. Participants with high numbers of solar keratoses, compared to people without solar keratoses, were most likely to experience the highest BCC counts overall (OR 3.3, 95%CI 1.4-13.5). Occurrences of BCC on the trunk (OR 3.3, 95%CI 1.4-7.6) and on the limbs (OR 3.7, 95%CI 2.0-7.0) were strongly associated with high numbers of solar keratoses on these sites. 4. Occurrence and determinants of basal cell carcinoma by histological subtype in an Australian community Among 1202 BCCs, 77% had a single growth pattern and 23% were of mixed histological composition. Among all BCCs the nodular followed by the superficial growth patterns were commonest. Risk of nodular and superficial BCCs on the head was raised if 5 or more solar keratoses were present on the face (OR 1.8, 95%CI 1.2-2.7 and OR 4.5, 95%CI 2.1-9.7 respectively) and similarly on the trunk in the presence of multiple solar keratoses on the trunk (OR 4.2, 95%CI 1.5-11.9 and OR 2.2, 95%CI 1.1-4.4 respectively). 5. Basal cell carcinoma and measures of cumulative sun exposure: an Australian longitudinal community-based study Dermal elastosis was more likely to be seen adjacent to head and neck BCCs than trunk BCCs (p=0.01). Severity of dermal elastosis increased on each site with increasing clinical signs of cutaneous sun damage on that site. BCCs that occurred without perilesional elastosis per se, were always found in an anatomic region with signs of photodamage. This thesis thus has identified the magnitude of the burden of multiple BCCs. It does not support the view that people affected by more than one BCC represent a distinct group of people who are prone to BCCs on certain body sites. The results also demonstrate that BCCs regardless of site, histology or order of occurrence are strongly associated with cumulative sun exposure causing photodamage to the skin, and hence challenge the view that BCCs occurring on body sites with typically low opportunities for sun exposure or of the superficial growth pattern are different in their association with the sun from those on typically sun-exposed sites, or nodular BCCs, respectively. Through dissemination in the scientific and medical literature, and to the community at large, these findings can ultimately assist in the primary and secondary prevention of BCC, perhaps especially in high-risk populations.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

Background Although PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents. Methods We have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2). Results 15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis. Conclusion Troglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

Background Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts. Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection. Results We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/ cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium. Conclusion We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Carcinoma invasion and metastasis : a role for epithelial-mesenchymal transition?

Carcinogenesis involves the accretion of unprogrammed genetic and epigenetic changes, which lead to dysregulation of the normal control of cell number. But a key clinical turning point in carcinoma progression is the establishment by emigrant cells of secondary growth sites (i.e., metastasis). The metastatic “cascade” comprises numerous steps, including escape from the primary tumor site, penetration of local stroma, entry of local vascular or lymphatic vessels (intravasation), aggregation with platelets, interaction with and adhesion to distant endothelia, extravasation, recolonization, and expansion ( 1), all the time avoiding effective immune clearance and being able to survive in these multiple contexts...

Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells

ConA-induced cell surface activation of pro-matrix metalloproteinase-2 (pro-MMP-2) by MDA-MB-231 human breast cancer cells is apparently mediated by up-regulation of membrane type 1 MMP (MT1-MMP) through transcriptional and posttranscriptional mechanisms. Here, we have explored the respective roles of cell surface clustering and protein tyrosine phosphorylation in the ConA- induction effects. Treatment with succinyl-ConA, a variant lacking significant clusterability, partially stimulated MT1-MMP mRNA and protein levels but did not induce MMP-2 activation, suggesting that clustering contributes to the transcriptional regulation by ConA but appears to be critical for the nontranscriptional component. We further found that genistein, an inhibitor of tyrosine phosphorylation, blocked ConA-induced pro-MMP-2 activation and ConA-induced MT1-MMP mRNA level in a dose-dependent manner, implicating tyrosine phosphorylation in the transcriptional aspect. This was confirmed by the dose-dependent promotion of pro-MMP-2 activation by sodium orthovanadate in the presence of suboptimal concentrations of ConA (7.5 μg/ml), with optimal effects seen at 25 μg/g orthovanadate. Genistein did not inhibit the ConA potentiation of MMP-2 activation in MCF-7 cells, in which transfected MT1-MMP is driven by a heterologous promoter, supporting the major implication of phosphotyrosine in the transcriptional component of ConA regulation. These data describe a major signaling event upstream of MT1- MMP induction by ConA and set the stage for further analysis of the nontranscriptional component.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.