Zainab Basir

Promoter Hypermethylation of Progesterone Receptor Isoform B (PR-B) in Endometriosis

The physiological effects of progesterone (P) is mediated by two isoforms of progesterone receptors (PRs): PR-A and PR-B. Progestins have long been used in the treatment of endometriosis but unfortunately the relief of pain is relatively short-term. In addition, about 9% of women with endometriosis simply do not respond to progestin therapy due to reasons unknown. In fact, a general tendency for relative progesterone resistance within eutopic and ectopic endometrium of women with endometriosis and the downregulation of PR-B, but not PR-A, in endometriosis have been noted. Since promoter hypermethylation is well-documented to be associated with transcriptional silencing, we sought to determine the methylation status of the PR-A and PR-B promoter regions in the epithelial component of endometriotic implants using a combination of laser capture microdissection (LCM), methylation specific PCR, and bisulfite sequencing. We found that the promoter region of PR-B, but not PR-A, is hypermethylated in endometriosis as compared with controls. In addition, the PR-B expression was significantly reduced in the ectopic endometrium. Our finding suggests that progesterone resistance in endometriosis in general and the down-regulation of PR-B, but not PR-A, in particular, are a result of promoter hypermethylation of PR-B, but not PR-A. This, in conjunction with our reported aberrant methylation of HOXA10 in the eutopic endometrium of women with endometriosis, strongly suggests that endometriosis is an epigenetic disease. This perspective should potentially open up new avenues for the delineation of pathogenesis of endometriosis, and might also lead to novel ways to treat the disease through reversing aberrant methylation via pharmacological means.

Resolution of clonal origins for endometriotic lesions using laser capture microdissection and the human androgen receptor (HUMARA) assay

OBJECTIVE To determine the clonal origins of endometriotic lesions using laser capture microdissection and PCR-based HUMARA assay. DESIGN Molecular genetic study of human tissue. SETTING Molecular genetics laboratory in an academic setting. PATIENT(S) Twenty patients with endometriosis. Forty specimens of endometriotic lesions from these patients and one specimen of normal endometrium were analyzed. INTERVENTION(S) Laser capture microdissection was used to harvest epithelial cells from single and multifocal endometrial lesions from paraffin-embedded and frozen tissues, and their clonality was determined with the HUMARA assay. MAIN OUTCOME MEASURE(S) Polymerase chain reaction-based HUMARA assay of clonality. RESULT(S) Thirty-eight specimens were polymorphic and thus informative. Most specimens were monoclonal, as determined by the HUMARA assay. In four specimens of multifocal lesions, polyclonality was detected, but upon more refined microdissections and further analyses, we found that each focus was monoclonal individually. CONCLUSION(S) Previously reported polyclonality is very likely to be attributed to the pooling of multifocal lesions or contamination of normal tissues. These results suggest that endometriotic lesions were monoclonal in origin, and in the case of multifocal lesions, each focus originates monoclonally; hence, different foci have independent origins. The monoclonality of endometriotic lesions suggests that they may carry neoplastic potentials, and the apparent independent origins of multifocal lesions suggest that reconstruction of individual lesion histories may help us to understand the initiation and progression of endometriosis.

p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38γ MAPK, and the activated c-Jun then recruits p38γ as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38γ that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38γ interacting with c-Jun. p38γ requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38γ are required for the trans-activation of MMP9. The active p38γ/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38γ and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response.

PTPH1 Dephosphorylates and Cooperates with p38γ MAPK to Increase Ras Oncogenesis through PDZ-Mediated Interaction

Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.

Genomic alterations in ectopic and eutopic endometria of women with endometriosis.

Background/Aims: Ectopic and eutopic endometria of women with endometriosis have been shown to contain genomic alterations. In this study, we sought to identify genomic alterations in both ectopic and eutopic endometria of 5 women with endometriosis and to examine whether the two tissues share any genomic alterations. We also attempted to classify tissue samples based on the alteration profiles. Methods: Laser capture microdissection was used to harvest epithelial cells. High-resolution comparative genomic hybridization microarrays were used to identify genomic alterations in eutopic and ectopic endometria from 5 women with endometriosis. The results were validated by real-time RT-PCR and loss of heterozygosity analysis. Results: All 5 patients had genomic alterations in their eutopic and ectopic endometria. The ectopic and eutopic endometria shared a sizable portion of genomic alterations. Cluster analysis of the genomic alteration profile correctly and consistently classified tissue samples from the 5 patients into two groups: peritoneal implants and ovarian cysts. Conclusions: The correct classification of tissue samples into two groups suggests that these two subtypes of endometriosis may have distinct genomic alteration profiles and are thus distinct entities, as previously proposed. The shared alterations are likely the ones that harbor genes responsible for an increased propensity of endometrial debris to implant to the ectopic sites and for early events that lead to the establishment of lesions. Alternatively, these shared alterations may harbor genes that are dysregulated in both eutopic and ectopic endometria. The identified genomic alterations should help to zero in genes involved in the pathogenesis of endometriosis in future studies.

Evaluation of crystals in formalin-fixed, paraffin-embedded tissue sections for the differential diagnosis of pseudogout, gout, and tumoral calcinosis.

Hematoxylin-eosin (H&E)–stained sections may not allow proper evaluation of birefringence properties of the crystals in the lesions of pseudogout, gout, and tumoral calcinosis. This study was undertaken to verify the application of a special stain that could facilitate the evaluation of the birefringence properties of these crystals for definitive diagnosis. We evaluated previously described nonaqueous alcoholic eosin staining (NAES) method based on the principle of using alcoholic eosin without hematoxylin and any other aqueous reagents for staining of formalin-fixed, paraffin-embedded tissue sections. Two observers, in a blinded fashion, evaluated the sections stained with routine H&E and NEAS method without the knowledge about clinical diagnosis. All pseudogout (nine sections from seven cases) and gout (eight sections from five cases) lesions demonstrated birefringence in the sections stained with NAES method. H&E–stained sections showing the respective diagnostic histomorphology failed to demonstrate the birefringent crystals by polarizing microscopy in all the eight sections from gout and in seven of nine sections from pseudogout. Only two H&E–stained sections showed scant calcium pyrophosphate dihydrate (CPPD) crystals in pseudogout. None of the three sections from two cases of tumoral calcinosis showed birefringence with either stain. We conclude that CPPD in pseudogout and monosodium urate in gout may not polarize in the routine H&E–stained sections. However, polarizing microscopy of sections stained with NAES method allowed demonstration of CPPD crystals with positive birefringence in pseudogout, MSU crystals with negative birefringence in gout, and calcium hydroxyapatite crystals without birefringence in tumoral calcinosis. Section stained with NAES method is a significantly useful adjunct to the routine H&E stain for proper evaluation of the crystals under polarizing microscope in these lesions.

PTPH1 cooperates with vitamin D receptor to stimulate breast cancer growth through their mutual stabilization

Tyrosine phosphorylation is tightly regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), and has a critical role in malignant transformation and progression. Although PTKs have a well-established role in regulating breast cancer growth, contribution of PTPs remains mostly unknown. Here, we report that the tyrosine phosphatase PTPH1 stimulates breast cancer growth through regulating vitamin D receptor (VDR) expression. PTPH1 was shown to be overexpressed in 49% of primary breast cancer and levels of its protein expression positively correlate with the clinic metastasis, suggesting its oncogenic activity. Indeed, PTPH1 promotes breast cancer growth by a mechanism independent of its phosphatase activity, but dependent of its stimulatory effect on the nuclear receptor VDR protein expression and depletion of induced VDR abolishes the PTPH1 oncogenic activity. Additional analyses showed that PTPH1 binds VDR and increases its cytoplasmic accumulation, leading to their mutual stabilization and stable expression of a nuclear localization-deficient VDR abolishes the growth-inhibitory activity of the receptor independent of 1,25-dihydroxyvitamin D3. These results reveal a new paradigm in which a PTP may stimulate breast cancer growth through increasing cytoplasmic translocation of a nuclear receptor, leading to their mutual stabilization.

Prostate-Specific Antigen Expression and Lipochrome Pigment Granules in the Differential Diagnosis of Prostatic Adenocarcinoma Versus Seminal Vesicle-Ejaculatory Duct Epithelium

BACKGROUND Lipochrome pigment granules (LPGs) and prostate-specific antigen (PSA) localization have been cited as helpful adjuncts in differentiating atypical histologic patterns of seminal vesicle-ejaculatory duct (SVED) from prostatic adenocarcinoma. However, LPGs have been described in both benign and neoplastic prostatic acini, and PSA expression within the intraprostatic SVED has not been fully explored. DESIGN Fifty radical prostatectomy specimens were studied for LPGs and 9 cases for PSA expression. RESULTS Two morphologic types of LPGs (type 1 and type 2) were observed. The reproducibility in classifying LPGs was evaluated by kappa statistics, which demonstrated a strong agreement between 4 observers. Type 1 was restricted to SVED in all 50 specimens. Type 2 was subclassified into 2A and 2B. Type 2 LPGs were observed in prostatic acini of different zones, high-grade prostatic intraepithelial neoplasia, prostatic adenocarcinoma, and occasionally with type 1 LPG in SVED. Focal reactivity for PSA in the distal portion of SVED near urethra was noted in 1 of 9 cases. CONCLUSION Awareness about morphologic differences between the 2 types of LPGs could help to avoid a potential diagnostic pitfall of misinterpreting SVED epithelium for adenocarcinoma. Caution is recommended in interpreting PSA expression, since rare focal PSA reactivity was observed in the distal SVED.

p38γ Mitogen-activated Protein Kinase (MAPK) Confers Breast Cancer Hormone Sensitivity by Switching Estrogen Receptor (ER) Signaling from Classical to Nonclassical Pathway via Stimulating ER Phosphorylation and c-Jun Transcription

Background: ER signals through binding to estrogen-responsive elements and interacting with c-Jun. Results: p38γ phosphorylates ER at Ser-118. This increases ER-c-Jun binding, promotes AP-1-dependent transcription, and confers breast cancer hormone sensitivity. Conclusion: p38γ increases breast cancer hormone sensitivity by regulating ER signaling between classical and nonclassical pathways. Significance: Regulating p38γ activity may be a new approach to increase breast cancer hormone sensitivity. Estrogen receptor (ER) α promotes breast cancer growth by regulating gene expression through classical estrogen response element (ERE) binding and nonclassical (interaction with c-Jun at AP-1 sites) pathways. ER is the target for anti-estrogens such as tamoxifen (TAM). However, the potential for classical versus nonclassical ER signaling to influence hormone sensitivity is not known. Moreover, anti-estrogens frequently activate several signaling cascades besides the target ER, and the implications of these “off-target” signaling events have not been explored. Here, we report that p38γ MAPK is selectively activated by treatment with TAM. This results in both phosphorylation of ER at Ser-118 and stimulation of c-Jun transcription, thus switching ER signaling from the classical to the nonclassical pathway leading to increased hormone sensitivity. Unexpectedly, phosphorylation at Ser-118 is required for ER to bind both p38γ and c-Jun, thereby promoting ER relocation from ERE to AP-1 promoter sites. Thus, ER/Ser-118 phosphorylation serves as a central mechanism by which p38γ regulates signaling transduction of ER with its inhibitor TAM.

Accuracy of Cytologic Interpretation of Pancreatic Neoplasms by Fine Needle Aspiration and Pancreatic Duct Brushings

OBJECTIVE To investigate the accuracy of fine needle aspiration (FNA) specimens and pancreatic duct brushings in the detection of pancreatic lesions and to compare the results with follow-up biopsy and/or surgical interpretation. STUDY DESIGN We reviewed a total of 57 specimens (37/20), 37 FNA specimens and 20 pancreatic duct brushings, from 45 patients treated at Froedtert Memorial Lutheran Hospital, affiliated with the Medical College of Wisconsin, Milwaukee, over a 4-year period. The FNA and brushing samples were categorized as follows: positive for malignancy (21/3 = 24), suspicious for malignancy (8/7 = 15) and atypical (8/10 = 18). The results were then correlated with the tissue diagnosis. RESULTS The 24 cytologic samples positive for malignancy included 23 (20/3) pancreatic ductal carcinoma (CA) and 1 (1/0) neuroendocrine CA; in the suspicious category, 11 (6/5) were pancreatic ductal CA; 2 (0/2) mucinous neoplasms and (2/0) neuroendocrine neoplasms; in the atypical category; 2 (2/0) suggestive of mucinous neoplasia, 1 (1/0) suggestive of serous neoplasia and 9 (2/7) favor reactive; and 6 (3/3) without further categorization. Tissue diagnoses were available in 26 cases: 12 (10/2) cases positive for malignancy, 8 (5/3) suspicious for malignancy and 6 (5/1) atypical. The 12 cytologically positive cases confirmed by histology showed 10 ductal CA, 1 neuroendocrine CA and 1 negative. All 8 cases (100%) suspicious for malignancy revealed positive results, including 5 ductal CA, 1 neuroendocrine neoplasm, 1 mucinous cystic neoplasm and 1 lymphoma. Of the 6 atypical lesions, 1 showed ductal CA, 2 mucinous cystic neoplasm and 3 chronic pancreatitis. CONCLUSION Pancreatic FNA and duct brushings [table: see text] are accurate methods in identifying pancreatic lesions, particularly ductal CA. Accuracy can be improved in the case of mucinous and other lesions with adequate cellularity of the smear and recognizing the limitations of brush samples in the case of mucinous cystic lesions. False negative results may occur in cases of poor representation of malignant cells or poor sampling.