Progression Tissue Microarray Research Articles

MST1R (RON) expression is a novel prognostic biomarker for metastatic progression in breast cancer patients.

This study evaluates the prognostic significance of MST1R (RON) expression in breast cancer with respect to disease progression, long-term survival, subtype, and association with conventional prognostic factors. The approach includes interrogation of survival and tumor staging with paired MST1R RNA expression from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Protein expression evaluation was performed using immunohistochemistry (IHC) staining of MST1R on breast cancer tissue samples from the Cancer Diagnosis Program Breast Cancer Progression tissue microarray and locally obtained breast tumor tissue samples analyzed with paired survival, metastasis, and subtype. Data from TCGA (n = 774) show poorer relapse-free survival (RFS) in patients with high MST1R expression (P = 0.32) and no difference in MST1R expression based on tumor stage (P = 0.77) or nodal status (P = 0.94). Patients in the GEO-derived Kaplan-Meier Plotter microarray dataset demonstrate the association of MST1R and poorer overall survival (n = 1402, P = 0.018) and RFS in patients receiving chemotherapy (n = 798, P = 0.041). Patients with high MST1R expression display worse overall survival (P = 0.01) and receiver operator characteristic (ROC) analysis demonstrate the predictive capacity of increased MST1R with early death (P = 0.0017) in IHC-stained samples. Paired IHC-stained breast tumor samples from the primary versus metastatic site show MST1R expression is associated with metastatic progression (P = 0.032), and ROC analysis supports the predictive capacity of MST1R in metastatic progression (P = 0.031). No associations of MST1R with estrogen receptor (ER), progesterone receptor (PR), both ER and PR, HER2 positivity, or triple-negativity were found (P = 0.386, P = 0.766, P = 0.746, P = 0.457, P = 0.947, respectively). MST1R expression has prognostic value in breast cancer with respect to survival and metastatic progression. MST1R expression is not associated with tumor stage, nodal status, or subtype.

Abstract 3498: Regulation of AR through an FGF5 driven mechanism

Abstract Purpose To investigate the role of increasing FGF5 expression in the progression of prostate cancer and emergence of castration resistant prostate cancer. Methods Utilizing a cell line model representing different stages of prostate cancer progression, we assessed FGF5 expression and function in vitro and in vivo. We analyzed both an outcomes and progression tissue microarray (TMA) containing ~400 prostate cancer patient cores to assess FGF5 expression in a clinical setting. Results There was a significant increase in the both RNA and protein expression of FGF5 in tumorigenic cell lines compared to nontumorigenic. In TMAs that were analyzed, there was a significant increase of FGF5-positive cells across progression of prostate cancer from benign to metastatic, with the highest percentage occurring in cases of cancer. When performing gain of function experiments with recombinant FGF5 (rFGF5) or overexpression plasmids, we found that FGF5 significantly increased the proliferation of AR-positive cell lines. Conversely, in AR-negative cell lines, FGF5 inhibited proliferation. Because FGF5 is associated with stem cells and differentiation events, we next asked whether the population of FGF5 cells that increases through cancer progression could lead to molecular changes in the total population. In addition to inhibiting proliferation of AR-negative nontumorigenic BPH1 cells, rFGF5 resulted in a robust AR expression. In LNCaP, C4-2, and C4-2B rFGF5 resulted in an increase in AR expression. In C4-2 and C4-2B cells an increase in AR protein coincided with an increase in nuclear YAP1, a potent transcriptional regulator. We hypothesized that androgens could be regulating FGF5 expression in the prostate. When androgen dependent LNCaP cells are subjected to an androgen-depleted environment, FGF5 protein and RNA significantly increased within 24hrs. Additionally, when AR is stimulated using dihyrotestosterone, FGF5 protein and RNA levels significantly decreased. These effects are not observed in cell lines that do not express AR. Conclusions These results suggest that FGF5 is increased in prostate cancer progression cell lines as well as patient samples, with highest expression in aggressive cancer cores. Data also suggests that AR plays a role in the regulation of FGF5 and vice versa. These data suggest that FGF5 driven AR expression could be driven by YAP1 nuclear localization. This mechanism will be further interrogated. Citation Format: Dalton McLean. Regulation of AR through an FGF5 driven mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3498.

ADAR1-mediated regulation of melanoma invasion

Melanoma cells use different migratory strategies to exit the primary tumor mass and invade surrounding and subsequently distant tissues. We reported previously that ADAR1 expression is downregulated in metastatic melanoma, thereby facilitating proliferation. Here we show that ADAR1 silencing enhances melanoma cell invasiveness and ITGB3 expression. The enhanced invasion is reversed when ITGB3 is blocked with antibodies. Re-expression of wild-type or catalytically inactive ADAR1 establishes this mechanism as independent of RNA editing. We demonstrate that ADAR1 controls ITGB3 expression both at the post-transcriptional and transcriptional levels, via miR-22 and PAX6 transcription factor, respectively. These are proven here as direct regulators of ITGB3 expression. miR-22 expression is controlled by ADAR1 via FOXD1 transcription factor. Clinical relevance is demonstrated in patient-paired progression tissue microarray using immunohistochemistry. The novel ADAR1-dependent and RNA-editing-independent regulation of invasion, mediated by ITGB3, strongly points to a central involvement of ADAR1 in cancer progression and metastasis.

Protocols for Tissue Microarrays in Prostate Cancer Studies.

Tissue microarray (TMA) technology is a method for high-throughput analysis of tissue biomarkers, commonly used in translational cancer research. TMAs allow performing a variety of in situ applications on hundreds of tissue samples simultaneously using the same protocols as for conventional slides. Thereby, precious material from patient samples remains largely preserved while costs in resources and time in laboratory processing decrease. Therefore, a TMA is a powerful tool to identify and study biomarkers that may have a potential diagnostic, prognostic, and predictive value. Depending on the research question, there are different types of TMAs, such as progression TMA, outcome TMA, and tumor heterogeneity TMA. Since the first introduction of the TMA method almost 20years ago, most laboratories used manual tissue arrayers for manufacturing. Nowadays, automatic or semiautomatic devices are commercially available, which largely facilitates the technical construction. However, preparatory work remains the most time-consuming part in preparing TMAs. This chapter focuses on issues involved in design and construction of prostate cancer TMAs.

Histopathological expression analysis of intercellular adhesion molecule 1 (ICAM-1) along development and progression of human melanoma.

Intercellular adhesion molecule 1 (ICAM-1) protein is an important adhesion molecule that facilitates metastasis on one hand, and on the other hand supports the immunological synapse necessary for T-cell mediated elimination. The expression pattern of ICAM-1 in melanoma was studied more than two decades ago, mainly in cell lines or in unmatched melanoma specimens. By using real time PCR we could not demonstrate a clear difference in ICAM-1 mRNA levels between primary melanocytes and primary cultures of metastatic melanoma. However, immunohistochemistry staining of progression tissue microarray comprised of samples of different disease stages derived from different patients, demonstrated a dramatic ICAM-1 upregulation particularly upon the transition from primary tumor to lymph node metastasis. There was no significant difference between lymph node and distant metastases. Importantly, these results were confirmed in an independent tissue microarray comprised of patient-paired specimens from progressive stages of the patient’s disease. These data indicate that ICAM-1 upregulation is required to initiate the lymphatic spread of melanoma (Stage III) but no further increase is associated with progression to remote organs (Stage IV).

Role of the EphB2 receptor in autophagy, apoptosis and invasion in human breast cancer cells

The Eph and Ephrin proteins, which constitute the largest family of receptor tyrosine kinases, are involved in normal tissue development and cancer progression. Here, we examined the expression and role of the B-type Eph receptor EphB2 in breast cancers. By immunohistochemistry using a progression tissue microarray of human clinical samples, we found EphB2 to be expressed in benign tissues, but strongly increased in cancers particularly in invasive and metastatic carcinomas. Subsequently, we found evidence that EphB2, whose expression varies in established cell breast lines, possesses multiple functions. First, the use of a DOX-inducible system to restore EphB2 function to low expressers resulted in decreased tumor growth in vitro and in vivo, while its siRNA-mediated silencing in high expressers increased growth. This function involves the onset of apoptotic death paralleled by caspases 3 and 9 activation. Second, EphB2 was also found to induce autophagy, as assessed by immunofluorescence and/or immunoblotting examination of the LC3, ATG5 and ATG12 markers. Third, EphB2 also has a pro-invasive function in breast cancer cells that involves the regulation of MMP2 and MMP9 metalloproteases and can be blocked by treatment with respective neutralizing antibodies. Furthermore, EphB2-induced invasion is kinase-dependent and is impeded in cells expressing a kinase-dead mutant EphB2. In summary, we identified a mechanism involving a triple role for EphB2 in breast cancer progression, whereby it regulates apoptosis, autophagy, and invasion.

Importance of glycolysis and oxidative phosphorylation in advanced melanoma.

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays

The Vectra platform (Caliper Life Sciences, Hopkinton, MA) is an advanced multispectral imaging system for biomarker quantitation in tissue microarray or intact tissue sections. This is the first study to validate its reliability for quantitating spatially overlapping biomarkers using chromogenic multiplexed immunohistochemistry on prostate tissue microarrays. Two tissue microarray cohorts (an outcome tissue microarray and a progression tissue microarray) were used. The outcome tissue microarray cohort consists of 462 duplicate cores with more than 5-year outcome information. The progression tissue microarray cohort consists of 384 duplicate cores from different disease (stage) groups. The tissue microarray slides were stained with different combinations of antibodies (anti-androgen receptor, anti-E-cadherin, anti-erythroblastosis virus E26 oncogene-related gene product, and anti-α-methylacyl-CoA racemase). Three outcome tissue microarrays were stained with androgen receptor + erythroblastosis virus E26 oncogene-related gene + E-cadherin (outcome tissue microarray 1), androgen receptor + E-cadherin (outcome tissue microarray 2), and erythroblastosis virus E26 oncogene-related gene + E-cadherin (outcome tissue microarray 3), respectively. One progression tissue microarray section was stained with E-cadherin and α-methylacyl-CoA racemase; tissue microarray slides were then scanned with the Vectra platform. Biomarker expression analysis was performed with Vectra software-Nuance 3.0.0, and inForm 1.2. IBM SPSS Statistics 19 was used for statistical and correlation analysis (SPSS, Chicago, IL). Close concordance was found between the triple- and double-immunostaining assays used for quantitating spatially overlapping biomarkers androgen receptor and erythroblastosis virus E26 oncogene-related gene using outcome tissue microarrays (r = 0.897 for androgen receptor and 0.613 for erythroblastosis virus E26 oncogene-related gene, respectively). α-Methylacyl-CoA racemase and E-cadherin expression levels measured in progression tissue microarray were consistent with previously published data by other groups. In conclusion, Vectra technology is reliable for objective and high-throughput biomarker quantitation and colocalization study using chromogenic multiplexed immunohistochemistry.

PAGE4 Positivity Is Associated with Attenuated AR Signaling and Predicts Patient Survival in Hormone-Naive Prostate Cancer

Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.

Negative Cross Talk between NFAT1 and Stat5 Signaling in Breast Cancer

The molecular mechanisms that modulate the activity of the signal transducers and activators of transcription 5 (Stat5) during the progression of breast cancer remain elusive. Here, we present evidence that the calcineurin/nuclear factor of activated T cells (NFAT) pathway negatively regulates the activation of Stat5, and vice versa in breast cancer. NFAT1 interacts with Stat5 in breast cancer cells, and their physical association is mediated by the DNA binding and transactivation domains of Stat5. Ectopically expressed NFAT1 is capable of inhibiting Stat5-dependent functions, including Stat5 transactivation, Stat5-mediated transcription of the downstream target gene expression, and binding of Stat5a to the Stat5 target promoter. By contrast, overexpression of a selective NFAT inhibitor VIVIT reversed NFAT1-mediated suppression of Stat5-dependent gene expression, whereas silencing of NFAT1 through RNA interference enhanced prolactin-induced, Stat5-mediated gene transcription, and breast cancer cell proliferation. A reciprocal inhibitory effect of Stat5 activity on NFAT1 signaling was also observed, implying these two signaling cascades antagonize each other in breast cancer. Importantly, analysis of a matched breast cancer progression tissue microarray revealed a negative correlation between levels of NFAT1 and Stat5 (pY694) during the progression of breast cancer. Taken together, these studies highlight a novel negative cross talk between the NFAT1- and Stat5-signaling cascades that may affect breast tumor formation, growth, and metastasis.

Higher Expression of the Heterogeneous Nuclear Ribonucleoprotein K in Melanoma

The heterogeneous nuclear ribonucleoprotein (hnRNP) K is an essential RNA and DNA binding protein involved in gene expression and signal transduction. The role of hnRNP K in cancer is relatively understudied. However, several cellular functions strongly indicate that hnRNP K is involved in tumorigenesis. Oncogenes c-Src, c-myc, and eIF4E are regulated by hnRNP K. We have shown an increased cytoplasmic hnRNP K in pancreatic cancer. In the present study, we investigated the altered expression of hnRNP K protein and its correlation with p-ERK in melanoma using human melanoma cell lines and tissue microarray. The protein levels of hnRNP K and p-ERK in 8 human melanoma cell lines and a melanoma progression tissue microarray containing 80 melanoma, 23 dysplastic nevi, and 14 benign nevi specimens were analyzed using Western blot and immunohistochemistry analysis. hnRNP K was knocked down by siRNA, and its effect on melanoma cells was assessed. We showed a higher hnRNP K protein level in both melanoma cell lines and melanoma tissue specimens, which correlated with a higher c-myc expression. An increase in the cytoplasmic hnRNP K and eIF4E protein levels in melanoma cells is also seen. p-ERK level was also higher in dysplastic nevi and melanoma tissues, but did not correlate with hnRNP K protein level. We then demonstrated that knocking down of hnRNP K by siRNA inhibited melanoma cell growth and colony formation, as well as c-myc expression. hnRNP K expression correlated with melanoma and may play a role in melanoma tumorigenesis.

Melanoma biomarker expression in melanocytic tumor progression: a tissue microarray study

The elucidation of protein biomarkers that are differentially expressed in human melanocytic tumors during tumor progression may lead to the identification of therapeutic targets and novel diagnostic tests. In a meeting chaired by Dr Mihm, a list of biomarkers of interest in melanoma was compiled. The specialized programs of research excellence (SPORE) in skin cancer developed a melanocytic tumor progression tissue microarray (TMA) to evaluate these candidate biomarkers. In addition to markers reported elsewhere, we evaluated c-Kit, MITF, MART1, HMB-45 and bcl-2. The TMA contains 480 cores of benign nevi, primary cutaneous melanoma and melanoma metastases. Immunohistochemical detection of melanoma biomarkers, including c-Kit, MITF, MART-1, HMB-45 and bcl-2 was performed. Intense nuclear staining for MITF protein was observed in 83% of nevi, 56% of primary melanomas and 23% of metastases. Bcl-2 expression was reduced with progression to metastasis (detected in 86, 89 and 52% of nevi, primaries and metastases, respectively), contrary to MART-1, which showed no differential expression (74, 85 and 84%). HMB-45 was observed in 18% of nevi and most (72 and 75%) primary melanomas and metastases. c-Kit protein increased with progression from nevi to primary tumor (10 and 77% of cases, respectively) and was decreased in metastases (26% of cases). Through a collaboration of the Skin SPOREs sponsored by the Organ Systems Branch of the National Cancer Institute (NCI), we identified a list of melanoma biomarkers of interest, developed a melanocytic tumor progression TMA and completed a coordinated analysis of these biomarkers. This TMA has served as a powerful validation tool for newly identified and known melanoma biomarkers by revealing trends in expression during tumor progression and by confirming the heterogeneity of biomarker expression in cutaneous melanocytic tumors.

A Novel Experimental Heme Oxygenase-1–Targeted Therapy for Hormone-Refractory Prostate Cancer

Heme oxygenase-1 (HO-1), a member of the heat shock protein family, plays a key role as a sensor and regulator of oxidative stress. Herein, we identify HO-1 as a biomarker and potential therapeutic target for advanced prostate cancer (PCA). Immunohistochemical analysis of prostate tissue using a progression tissue microarray from patients with localized PCA and across several stages of disease progression revealed a significant elevation of HO-1 expression in cancer epithelial cells, but not in surrounding stromal cells, from hormone-refractory PCA (HRPCA) compared with hormone-responsive PCA and benign tissue. Silencing the ho-1 gene in HRPCA cells decreased the HO-1 activity, oxidative stress, and activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase/p38 kinase. This coincided with reduced cell proliferation, cell survival, and cell invasion in vitro, as well as inhibition of prostate tumor growth and lymph node and lung metastases in vivo. The effect of ho-1 silencing on these oncogenic features was mimicked by exposure of cells to a novel selective small-molecule HO-1 inhibitor referred to as OB-24. OB-24 selectively inhibited HO-1 activity in PCA cells, which correlated with a reduction of protein carbonylation and reactive oxygen species formation. Moreover, OB-24 significantly inhibited cell proliferation in vitro and tumor growth and lymph node/lung metastases in vivo. A potent synergistic activity was observed when OB-24 was combined with Taxol. Together, these results establish HO-1 as a potential therapeutic target for advanced PCA.

Expression of Cyclophilin B is Associated with Malignant Progression and Regulation of Genes Implicated in the Pathogenesis of Breast Cancer

Cyclophilin B (CypB) is a 21-kDa protein with peptidyl-prolyl cis-trans isomerase activity that functions as a transcriptional inducer for Stat5 and as a ligand for CD147. To better understand the global function of CypB in breast cancer, T47D cells with a small interfering RNA-mediated knockdown of CypB were generated. Subsequent expression profiling analysis showed that 663 transcripts were regulated by CypB knockdown, and that many of these gene products contributed to cell proliferation, cell motility, and tumorigenesis. Real-time PCR confirmed that STMN3, S100A4, S100A6, c-Myb, estrogen receptor alpha, growth hormone receptor, and progesterone receptor were all down-regulated in si-CypB cells. A linkage analysis of these array data to protein networks resulted in the identification of 27 different protein networks that were impacted by CypB knockdown. Functional assays demonstrated that CypB knockdown also decreased cell growth, proliferation, and motility. Immunohistochemical and immunofluorescent analyses of a matched breast cancer progression tissue microarray that was labeled with an anti-CypB antibody demonstrated a highly significant increase in CypB protein levels as a function of breast cancer progression. Taken together, these results suggest that the enhanced expression of CypB in malignant breast epithelium may contribute to the pathogenesis of this disease through its regulation of the expression of hormone receptors and gene products that are involved in cell proliferation and motility.

Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

IntroductionMammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.MethodsExpression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L).ResultsWe found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding α2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes.ConclusionCollectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.

The biological significance of FHIT protein expression in lung cancer and precancerous tissues detected by tissue microarray

Fragile histidine triad (FHIT) is a candidate tumor suppressor gene. Aberrant expression of FHIT has been observed in multiple carcinomas induced by environmental carcinogens, especially in lung cancer. In this study, the expression of FHIT protein in lung cancer progression tissue microarray was detected and their roles in oncogenesis and progression of lung cancer were discussed. The expression of FHIT protein in tissue microarray with 270 cores was detected by SP immunohistochemistry method, in which there were 89 cases of primary lung cancer, 12 cases of lymph node metastasis of lung cancer, 12 cases of precancerous lesion and 10 cases of normal lung tissue, and the clinicopathological features of lung cancer were analyzed. The expression of FHIT was localized in the cytoplasm. Loss of FHIT expression in primary cancers, precancerous lesion and lymph node metastasis of lung cancer was 46.1%, 41.7% and 50.0% respectively, while 0 in 10 cases of normal tissues. A significant difference of FHIT expression was observed among four groups (P < 0.05). Loss of FHIT expression in precancerous lesion, primary lung cancer and lymph node metastasis of lung cancer was significantly higher than that in normal lung tissue (P < 0.05). The difference among precancerous lesion, primary lung cancer and lymph node metastasis of lung cancer groups was not statistically significant (P > 0.05). Loss of FHIT expression was related to tumor histologicol types, degree of cell differentiation and the smoking history of patients (P < 0.05), but not to sex, age, gross appearance types, TNM stages, or lymph node metastasis (P > 0.05). The protein expression level of FHIT is reduced in primary cancers and precancerous tissues, especially in most squamous cell carcinomas, poorly differentiated group and the patients with a smoking history. These results indicate that loss of FHIT expression might correlate with carcinogenesis, development of lung cancer and the carcinogenesis induced by smoking.

Clinical Validation of Breast Cancer Biomarkers Using Tissue Microarray Technology

The results of previous studies done in our laboratory on breast cancer gene expression profile, using DNA microarrays, led to the discovery of several genes associated with breast cancer progression. Further evaluation of these genes and their involvement at various stages of cancer progression required performance of immunohistochemistry on thousands of different tissue blocks. Tissue microarray (TMA) technology facilitates rapid translation of DNA microarrays results to clinical specimens by using immunohistochemical analysis of protein expression. DNA microarray analysis done in our laboratory showed a significantly higher expression of prostatic-specific antigen (PSA) in invasive ductal carcinomas as compared to ductal carcinoma in situ, a finding contrary to the previously published data for PSA immunoreactivity in breast carcinomas. To find out whether TMA strategy could be used to explore the expression of the candidate genes involved in the breast cancer progression, we constructed a breast cancer progression TMA. It consisted of 2 normal ductal epithelium, 8 ductal carcinoma in situ, 19 invasive ductal carcinomas, and 3 metastatic ductal carcinomas of breast in triplets. Two prostatic adenocarcinomas and 2 normal colons were used as positive and negative controls, respectively. We first used well-documented and well-tested markers, such as antibodies to estrogen receptor, progesterone receptor, and p53. Results of these 3 antibodies were according to the previously published data. To validate our result, we then used antibody to PSA and looked for the expression of this protein on breast cancer progression TMA. Except for the 2 positive controls all 98 cores were found to be negative for PSA expression highlighting the importance of validation studies for DNA microarray results.

PEA-15 Inhibits Tumor Cell Invasion by Binding to Extracellular Signal-Regulated Kinase 1/2

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) binds to extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases to alter ERK1/2 cellular localization and target preferences and binds to adaptors in the extrinsic cell death pathway to block apoptosis. Here, we report that PEA-15 protein expression is inversely correlated with the invasive behavior of breast cancer in an immunohistochemical analysis of a breast cancer progression tissue microarray. Short hairpin RNA-mediated inhibition of PEA-15 expression increased the invasion of PEA-15-expressing tumor cells in vitro, suggesting a causative role for PEA-15 in the inhibition of invasion. This causative role was confirmed by the finding that the enforced expression of PEA-15 in invasive tumor cells reduced invasion. The effect of PEA-15 on tumor invasion is mediated by its interaction with ERK1/2 as shown by the following: (a) PEA-15 mutants that fail to bind ERK1/2 did not inhibit invasion; (b) overexpression of ERK1 or activated MAP/ERK kinase (MEK) reversed the inhibitory effect of PEA-15; (c) when an inhibitor of ERK1/2 activation reduced invasion, PEA-15 expression did not significantly reduce invasion further. Furthermore, we find that the effect of PEA-15 on invasion seems to relate to the nuclear localization of activated ERK1/2. PEA-15 inhibits invasion by keeping ERK out of the nucleus, as a PEA-15 mutant that cannot prevent ERK nuclear localization was not able to inhibit invasion. In addition, membrane-localized ERK1, which sequesters endogenous ERK1 to prevent its nuclear localization, also inhibited invasion. These results reveal that PEA-15 regulates cancer cell invasion via its ability to bind ERK1/2 and indicate that nuclear entry of ERK1/2 is important in tumor behavior.

Expression and Significance of Survivin mRNA in Lung Cancer of Different Progression Stages by FISH and Tissue Microarray Technology

Objective: To investigate the expression of Survivin mRNA in lung cancer progression tissue microarray by FISH (fluorescence in situ hybridization) method and determine its role and significance in lung cancer genesis and progress. Methods: The expression of Survivin mRNA was detected by FISH method and tissue microarray technology. 89 cases of primary lung cancer, 12 cases of lymph node metastasis of lung cancer, 12 cases of precancerous lesion and 10 cases of normal lung tissue were examined. Results: 69.7% of primary lung cancer express Survivin mRNA; the positive ratio of primary lung cancer and precancerous lesion were both significantly higher than that of normal lung tissue (P<0.05); the expression of Survivin mRNA was related to the differentiation degree, lymph node metastasis and clinical stages (P<0.05). Conclusion: FISH has good sensitivity and stability. Tissue microarray technology has many advantages, such as high efficiency, high throughput, etc; it may have good prospect in pathology. Survivin mRNA was highly expressed in lung cancer and precancerous lesion; it was related to the progress and malignant behavior; it may play a promotion role in lung cancer genesis and progress and offer basis to early diagnosis, prognosis estimate and treatment.

Expression of survivin, PTEN and BFGF in lung cancer progression tissue microarray

To investigate the expressions of survivin, PTEN and bFGF in lung cancer and precancerous lesion by tissue microarray technology. Methods: The expressions of Survivin, PTEN and bFGF were detected in 89 primary lung cancers, 12 lung cancers with lymph node metastasis, 12 precancerous lesions of lung by an immunohistochemical method, and 10 normal lung tissues were used as controls. Results: The expression of Survivin and bFGF protein were 57.3% and 66.3% in 89 primary lung cancer respectively, significantly higher than the control group (P<0.05). The positive ratio of PTEN was 42.7% in primary lung cancer, significantly lower than that of nornmal tissue (90.0%)(P<0.05). The expressions of Survivin and bFGF were significantly related to lymph node metastasis and clinical stages. The expression of PTEN was related to differentiations, lymph node metastasis and clinical stages (P<0.05). There was a negative correlation between expressions of Survivin and PTEN, a positive correlation between expressions of Survivin and bGFG (P<0.01). Conclusion: Survivin, PTEN and bFGF proteins may be related to the pathogenesis, progression and malignant behavious of lung cancer, and there are some relationships between expressions of Survivin, PTEN and bFGF. Survivin, PTEN and bFGF may play a role in prognosis assessment of lung cancer.