Integrins and Epithelial-Mesenchymal Cooperation in the Tumor Microenvironment of Muscle-Invasive Lethal Cancers.

Muscle-invasive lethal carcinomas traverse into and through this specialized biophysical and growth factor enriched microenvironment. We will highlight cancers that originate in organs surrounded by smooth muscle, which presents a barrier to dissemination, including prostate, bladder, esophageal, gastric, and colorectal cancers. We propose that the heterogeneity of cell-cell and cell-ECM adhesion receptors is an important driver of aggressive tumor networks with functional consequences for progression. Phenotype heterogeneity of the tumor provides a biophysical advantage for tumor network invasion through the tensile muscle and survival of the tumor network. We hypothesize that a functional epithelial-mesenchymal cooperation (EMC)exists within the tumor invasive network to facilitate tumor escape from the primary organ, invasion and traversing of muscle, and navigation to metastatic sites. Cooperation between specific epithelial cells within the tumor and stromal (mesenchymal) cells interacting with the tumor is illustrated using the examples of laminin-binding adhesion molecules-especially integrins-and their response to growth and inflammatory factors in the tumor microenvironment. The cooperation between cell-cell (E-cadherin, CDH1) and cell-ECM (α6 integrin, CD49f) expression and growth factor receptors is highlighted within poorly differentiated human tumors associated with aggressive disease. Cancer-associated fibroblasts are examined for their role in the tumor microenvironment in generating and organizing various growth factors. Cellular structural proteins are potential utility markers for future spatial profiling studies. We also examine the special characteristics of the smooth muscle microenvironment and how invasion by a primary tumor can alter this environment and contribute to tumor escape via cooperation between epithelial and stromal cells. This cooperative state allows the heterogenous tumor clusters to be shaped by various growth factors, co-opt or evade immune system response, adapt from hypoxic to normoxic conditions, adjust to varying energy sources, and survive radiation and chemotherapeutic interventions. Understanding the epithelial-mesenchymal cooperation in early tumor invasive networks holds potential for both identifying early biomarkers of the aggressive transition and identification of novel agents to prevent the epithelial-mesenchymal cooperation phenotype. Epithelial-mesenchymal cooperation is likely to unveil new tumor subtypes to aid in selection of appropriate therapeutic strategies.

Glyoxalase 1 Expression as a Novel Diagnostic Marker of High-Grade Prostatic Intraepithelial Neoplasia in Prostate Cancer.

Glyoxalase 1 (GLO1) is an enzyme involved in the detoxification of methylglyoxal (MG), a reactive oncometabolite formed in the context of energy metabolism as a result of high glycolytic flux. Prior clinical evidence has documented GLO1 upregulation in various tumor types including prostate cancer (PCa). However, GLO1 expression has not been explored in the context of PCa progression with a focus on high-grade prostatic intraepithelial neoplasia (HGPIN), a frequent precursor to invasive cancer. Here, we have evaluated GLO1 expression by immunohistochemistry in archival tumor samples from 187 PCa patients (stage 2 and 3). Immunohistochemical analysis revealed GLO1 upregulation during tumor progression, observable in HGPIN and PCa versus normal prostatic tissue. GLO1 upregulation was identified as a novel hallmark of HGPIN lesions, displaying the highest staining intensity in all clinical patient specimens. GLO1 expression correlated with intermediate-high risk Gleason grade but not with patient age, biochemical recurrence, or pathological stage. Our data identify upregulated GLO1 expression as a molecular hallmark of HGPIN lesions detectable by immunohistochemical analysis. Since current pathological assessment of HGPIN status solely depends on morphological features, GLO1 may serve as a novel diagnostic marker that identifies this precancerous lesion.

Laminin-5 beta3A expression in LNCaP human prostate carcinoma cells increases cell migration and tumorigenicity.

Interactions between extracellular matrix proteins and prostate carcinoma cells change dramatically during prostate tumor progression. We have concentrated on two key modifications that occur in the hemidesmosome in prostate carcinoma: loss of laminin-5 protein expression and altered basal cell polarity of the alpha6beta4 integrin. We previously demonstrated two cell line-specific isoforms (beta3A and beta3B) of the LAMB3 message. Cells expressing only the beta3B isoform did not translate the beta3 protein and were unable to assemble the laminin-5 trimer. One such cell line, LNCaP, was selected to determine whether restoration of the laminin-5 beta3A isoform would cause expression of a functional laminin-5 beta3 chain, assembly and secretion of the laminin-5 trimer, and reversion to a non-neoplastic phenotype. Laminin-5 beta3A cDNA was cloned and stably transfected into LNCaP cells. We observed the restoration of the beta3 protein, but a laminin-5 trimer was not secreted. Moreover, increased cell migration was demonstrated, and tumorigenicity was increased in SCID mice. A microarray analysis, performed between transfected and nontransfected LNCaP cells, showed most changing genes to be associated with signal transduction. The beta3 chain of laminin-5 may thus play an important role in signal transduction, which may enhance cell motility and tumorigenesis.

Cell line-specific translation of two laminin 5 beta3 chain isoforms.

In sequencing the beta3 chain of laminin 5 mRNA from LNCaP cells, we observed three different human cDNA clones (XM_001716, NM_000228 and L25541) in the GenBank that identified different sequences in the untranslated regions (UTR). XM_001716 and NM_000228 are almost identical cDNA clones with approximately 99% homology. However, they are quite different from L25541 in both the 5' UTR and the 3' UTR. Development of a PCR assay to specifically detect two of these different forms of the message led to the observation that they were differentially expressed in various cell lines. The message designated B3A (NM_000228, and XM_001716) was absent in LNCaP and MCF7 and greatly reduced in PC3-N, but was present in eight other epithelial cell lines. B3B (L25541) was present in all cell lines studied. The cell lines that failed to express the B3A form also failed to express the protein based on both immunoblotting and immunohistochemical analysis. It appears from this data that there are two isoforms of the beta3 mRNA, and that the 5' UTRs of the mRNAs play an important role in regulating translation of the beta3 protein. Since laminin 5 is lost in prostate carcinoma, the mechanism of control that results in the translation of the two forms of message may be important in tumorigenesis.

Immunohistochemical quantification of the vitamin B12 transport protein (TCII), cell surface receptor (TCII-R) and Ki-67 in human tumor xenografts.

BACKGROUND/AIM: Cancer cells have an essential demand for vitamin B12 (cobalamin) to enable cellular replication. The present pilot study quantified the immunohistochemical expression of vitamin B12 transport protein (Transcobalamin II; TCII), cell surface receptor (Transcobalamin II-R; TCII-R) and proliferation protein (Ki-67) in human tumor xenografts. MATERIALS AND METHODS: Tissue microarray slides containing 34 xenograft tumor tissues were immunohistochemically stained using TCN2 (anti-TCII), CD320 (anti-TCII-R) and MIB-1 (anti-Ki-67) antibodies. Representatively stained areas of all slides were digitally imaged and protein expression was quantified using ImageJ software plugins. RESULTS: All xenograft tumor tissues stained positively for TCII, TCII-R and Ki-67 proteins; expression varied both within and between tumor types. Correlation between TCII/TCII-R and Ki-67 expression was not significant in xenograft tissues. CONCLUSION: Proliferating cancer cells express measurable levels of TCII and TCII-R. Immunohistochemical quantification of these markers may be useful as a tool for detection of tumors, tailored selection of anti-tumor therapies and surveillance for evidence of recurrent disease.

CD44 Promotes Epithelial Mammary Gland Development and Exhibits Altered Localization during Cancer Progression.

The basal cell layer has emerged as a critical player in cancer progression, and understanding the molecular contribution of specific cell types is important in treatment and prevention. The adhesion receptor CD44, which mediates epithelial-stromal and cell-cell interactions, has been shown to both promote and suppress tumor progression. To better understand the normal function of CD44, we have investigated its role in mouse mammary gland development and its expression in human breast and prostate cancer. We have found that CD44 is expressed in the myoepithelium of the developing mammary gland and modulates ductal development of FVB/N mice. The loss of CD44 results in defective luminal-myoepithelial cell-cell adhesion and promotes the mixing of luminal and myoepithelial layers, disrupting epithelial bilayer organization, and CD44-null mice experience delayed ductal outgrowth and impaired terminal end bud formation. The myoepithelial expression of CD44 is also relevant to its expression in cancer, as CD44 is expressed in the basal cells of early-stage breast and prostate cancer but exhibits altered localization with increasing tumorigenicity and is strongly expressed by tumor epithelium.

Extracellular engagement of alpha6 integrin inhibited urokinase-type plasminogen activator-mediated cleavage and delayed human prostate bone metastasis.

Expression of alpha(6) integrin, a laminin receptor, on tumor cell surfaces is associated with reduced patient survival and increased metastasis in a variety of tumors. In prostate cancer, tumor extracapsular escape occurs in part via laminin-coated nerves and vascular dissemination, resulting in clinically significant bone metastases. We previously identified a novel form of alpha(6) integrin, called alpha(6)p, generated by urokinase-type plasminogen activator-dependent cleavage of the laminin-binding domain from the tumor cell surface. Cleavage increased laminin-dependent migration. Currently, we used the known conformation sensitivity of integrin function to determine if engagement of the extracellular domain inhibited integrin cleavage and the extravasation step of metastasis. We show that alpha(6) integrin was present on prostate carcinoma escaping the gland via nerves. Both endogenous and inducible levels of alpha(6)p were inhibited by engaging the extracellular domain of alpha(6) with monoclonal antibody J8H. J8H inhibited tumor cell invasion through Matrigel. A severe combined immunodeficient mouse model of extravasation and bone metastasis produced detectable, progressive osteolytic lesions within 3 weeks of intracardiac injections. Injection of tumor cells, pretreated with J8H, delayed the appearance of metastases. Validation of the alpha(6) cleavage effect on extravasation was confirmed through a genetic approach using tumor cells transfected with uncleavable alpha(6) integrin. Uncleavable alpha(6) integrin significantly delayed the onset and progression of osseous metastases out to six weeks post-injection. The results suggest that alpha(6) integrin cleavage permits extravasation of human prostate cancer cells from circulation to bone and can be manipulated to prevent metastasis.

Role of the extracellular matrix in prostate carcinogenesis.

This review summarizes the current state of knowledge regarding the proteins composing the extracellular matrix in the human prostate. The normal expression as well as the changes which occur in PIN and carcinoma are described for the lamins, collagens, and glycosaminoglycans.