[show abstract][hide abstract] ABSTRACT: FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2-31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2-31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2-31 significantly inhibited cell proliferation and viability (range 0.05-10 μM). More importantly, 15 mg/kg of INT2-31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2-31) has potential anti-neoplastic therapeutic effects in human melanoma.
[show abstract][hide abstract] ABSTRACT: Previously, we have shown that FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and, thereby, activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function have shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. In this study, as a novel approach in human melanoma, we evaluated the effect of a small molecule compound that disrupts the interaction of FAK and IGF-1R. Using virtual screening and functional testing, we identified a lead compound (INT2-31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, induce apoptosis, and decrease tumor growth in vivo. Based on GST pulldowns with purified protein fragments of FAK and IGF-1R and from co-immunoprecipitation assays from melanoma cells, INT2-31 blocked the interaction of FAK and IGF-1R and precluded activation of IRS-1 leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2-31 significantly inhibited cell proliferation and viability (range 0.05-10μM). More importantly, 15mg/kg of INT2-31 given for 21 days via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT resulting in significant melanoma tumor regression in vivo (p<0.05). Our data suggest that FAK-IGF-1R protein interaction can be identified as an important target and disruption of this interaction with a small molecule has potential anti-neoplastic therapeutic effects in human melanoma.
[show abstract][hide abstract] ABSTRACT: Deregulation of insulin-like growth factor-1 receptor (IGF-1R) and focal adhesion kinase (FAK) signaling pathways plays an important role in cancer cell proliferation and metastasis. In pancreatic cancer cells, the crosstalk and compensatory mechanisms between these two pathways reduce the efficacy of the treatments that target only one of the pathways. Ablation of IGF-1R signaling by siRNA showed minimal effects on the survival and growth of pancreatic cancer cells. An increased activity of FAK pathway was seen in these cells after IGF-1R knockdown. Further inhibition of FAK pathway using Y15 significantly decreased cell survival, adhesion, and promoted apoptosis. The combination of Y15 treatment and IGF-1R knockdown also showed significant antitumor effect in vivo. The current study demonstrates the importance of dual inhibition of both these signaling pathways as a novel strategy to decrease both in vitro and in vivo growth of human pancreatic cancer.
[show abstract][hide abstract] ABSTRACT: Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is overexpressed in many types of tumors, including pancreatic cancer, and plays an important role in cell adhesion and survival signaling. Pancreatic cancer is a lethal disease and is very resistant to chemotherapy, and FAK has been shown recently to assist in tumor cell survival. Therefore, FAK is an excellent potential target for anti-cancer therapy. We identified a novel small molecule inhibitor (1,2,4,5-Benzenetetraamine tetrahydrochloride, that we called Y15) targeting the main autophosphorylation site of FAK and hypothesized that it would be an effective treatment strategy against human pancreatic cancer. Y15 specifically blocked phosphorylation of Y397-FAK and total phosphorylation of FAK. It directly inhibited FAK autophosphorylation in a dose- and time-dependent manner. Furthermore, Y15 increased pancreatic cancer cell detachment and inhibited cell adhesion in a dose-dependent manner. Y15 effectively caused human pancreatic tumor regression in vivo, when administered alone and its effects were synergistic with gemcitabine chemotherapy. This was accompanied by a decrease in Y397-phosphorylation of FAK in the tumors treated with Y15. Thus, targeting the Y397 site of FAK in pancreatic cancer with the small molecule inhibitor, 1,2,4,5-Benzenetetraamine tetrahydrochloride, is a potentially effective treatment strategy in this deadly disease.
[show abstract][hide abstract] ABSTRACT: Vascular endothelial growth factor receptor-3 is a receptor tyrosine kinase that is overexpressed in some human carcinomas, but its role in tumorigenesis has not been fully elucidated. We examined VEGFR-3 expression in normal, nonneoplastic and early stage malignant breast tissues and have shown that VEGFR-3 upregulation in breast cancer preceded tumor cell invasion, suggesting that VEGFR-3 may function as a survival signal. We characterized the biological effects of VEGFR-3 over-expression in human breast cancer cells based on two approaches: gain of function by overexpressing VEGFR-3 in MCF-7 breast cancer cells and loss of function by RNAi-mediated silencing of VEGFR-3 in MCF-7-VEGFR-3 and BT474 cells. VEGFR-3 overexpression increased cellular proliferation by 40% when MCF7-VEGFR-3 cells were compared to parental MCF7 cells, and proliferation was reduced by more than 40% when endogenous VEGFR-3 was downregulated in BT474 cells. VEGFR-3 overexpression promoted a three-fold increase in motility and invasion and both motility and invasion were inhibited by downregulation of VEGFR-3. Furthermore, VEGFR-3 overexpression promoted cellular survival under stress conditions induced by staurosporine treatment and led to anchorage-independent growth. VEGFR-3 overexpression dramatically increased tumor formation in both hormone-dependent and independent xenograft models. With estrogen stimulation, MCF7-VEGFR-3 xenografts were ten times larger than control xenografts. Finally, downregulation of VEGFR-3 expression in both xenograft model cell lines led to a significant reduction of tumor growth. For the first time, we have demonstrated that VEGFR-3 overexpression promotes breast cancer cell proliferation, motility, survival, anchorage-independent growth and tumorogenicity in the absence of ligand expression.
[show abstract][hide abstract] ABSTRACT: Attenuation of the p53 protein is one of the most common abnormalities in human tumors. Another important marker of tumorigenesis is focal adhesion kinase (FAK), a 125-kDa tyrosine kinase that is overexpressed at the mRNA and protein levels in a variety of human tumors. FAK is a critical regulator of adhesion, motility, metastasis, and survival signaling. We have characterized the FAK promoter and demonstrated that p53 can inhibit the FAK promoter activity in vitro. In the present study, we showed that p53 can bind the FAK promoter-chromatin region in vivo by chromatin immunoprecipitation (ChIP) assay. Furthermore, we demonstrated down-regulation of FAK mRNA and protein levels by adenoviral overexpression of p53. We introduced plasmids with different mutations in the DNA-binding domain of p53 (R175H, p53 R248W and R273H) into HCTp53(-/-) cells and showed that these mutations of p53 did not bind FAK promoter and did not inhibit FAK promoter activity, unlike wild type p53. We analyzed primary breast and colon cancers for p53 mutations and FAK expression, and showed that FAK expression was increased in tumors containing mutations of p53 compared to tumors with wild type p53. In addition, tumor-derived missense mutations in the DNA-binding domain (R282, R249, and V173) also led to increased FAK promoter activity. Thus, the present data show that p53 can regulate FAK expression during tumorigenesis.
[show abstract][hide abstract] ABSTRACT: Early invasive vulvar squamous cell carcinoma (SCC) with less than 1.0 mm of invasion (FIGO stage IA) has been shown to have a minimal risk of lymph node metastasis and is associated with an excellent prognosis. The prognostic significance of other histologic parameters other than depth of invasion, however, remains controversial. Seventy-eight consecutive cases of vulvar SCC having a depth of invasion of 5.0 mm or less were reviewed and the clinical outcome compared with the type of surgical excision, the presence of concurrent lymph node metastases, the depth of tumor invasion, the tumor thickness, the tumor horizontal spread, the estimated tumor volume, tumor histologic subtype, tumor histologic grade, tumor pattern of invasion, tumor multifocality, presence of perineural invasion, presence of angiolymphatic invasion and the presence of precursor lesions, including the type of vulvar intraepithelial neoplasia and presence of lichen sclerosus. The only histologic feature for predicting concurrent lymph node metastasis was tumor depth of invasion. The 3 most important features of stage IA tumors in predicting tumor recurrence were the depth of invasion, presence of SCC at the surgical margins, and the histologic grade.
The American journal of surgical pathology 06/2008; 32(5):765-72. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: The protein p16(Ink4a) is overexpressed in cervical lesions associated with high-risk human papillomavirus (HPV) subtypes 16 and 18, but not in low-risk HPV subtypes 6 and 11 or non-HPV-associated cervical lesions.
To determine whether p16(Ink4a) expression in equivocal cervical lesions helps distinguish atypical non-HPV changes from HPV-related changes.
One hundred ninety-one cervical lesions, including 81 cervical intraepithelial neoplasia 1, 52 squamous metaplasia, 33 cellular features suggestive of HPV-related change, 9 reserve cell hyperplasia, 4 microglandular hyperplasia, and 12 inflammatory cervicitis, were randomly selected from archival cervical biopsy specimens. All 191 samples were studied with p16(Ink4a) (JC8 monoclonal antibody). Reactivity for p16(Ink4a) was scored on a 3-tier system as follows: negative, 0% to 5% cells reactive; focal/scattered positive, greater than 5% and less than or equal to 80% cells reactive; diffuse positive, greater than 80% cells reactive. Reactivity was based on normal/reactive cervical specimens where anti-p16 antibody was negative/weakly expressed in non-cervical epithelial cells. Cervical intraepithelial neoplasia 1 lesions not reactive for p16(Ink4a) were investigated for the presence of high-risk HPV by real-time polymerase chain reaction.
No p16(Ink4a) reactivity was detected in the cervical lesions associated with atypical non-HPV change. Eleven of the cervical intraepithelial neoplasia 1 lesions showed focal/scattered reactivity expression for p16(Ink4a), and 19 of the CIN 1 lesions had diffuse reactivity. Fifty of 51 of the CIN 1 lesions negative for p16(Ink4a) were real-time polymerase chain reaction negative for the presence of high-risk HPV; 1 was real-time polymerase chain reaction positive for high-risk HPV.
The data support the routine use of p16(Ink4a) immunohistochemical evaluation of cervical biopsy specimens for better discrimination of non-HPV-associated lesions from HPV-related lesions.
Archives of pathology & laboratory medicine 06/2008; 132(5):795-9. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: The focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase important in signaling between cells and their extracellular matrix. Studies have shown that FAK expression is up-regulated in several human tumors and is related to tumor progression. We recently found an increase in p125(FAK) expression in human neuroblastoma cells lines and wished to determine its expression in human neuroblastoma specimens and evaluate for a possible correlation between p125(FAK) expression and known prognostic factors for neuroblastoma. We hypothesized that p125(FAK) expression would be up-regulated in advanced human neuroblastomas.
Using immunohistochemical techniques with monoclonal antibody 4.47 specific for p125(FAK) expression, we analyzed 70 formalin-fixed, paraffin-embedded human neuroblastoma specimens for p125(FAK) staining. In addition, real-time PCR was used to determine the abundance of FAK mRNA in 17 matched human neuroblastoma mRNA specimens.
FAK staining was present in 51 of the 70 tumor specimens (73%). Immunohistochemical staining of p125(FAK) in the ganglion-type tumor cells correlated with advanced International Neuroblastoma Staging System tumor stages and FAK mRNA abundance. In addition, p125(FAK) staining was significantly increased in stage IV tumors with amplification of the N-MYC oncogene.
These novel findings provide evidence that FAK is expressed by advanced-stage neuroblastoma and provide a rationale for targeting FAK in the treatment of this tumor.
Clinical Cancer Research 06/2008; 14(11):3299-305. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Idiopathic granulomatous mastitis, also known as idiopathic granulomatous lobular mastitis, is a benign breast lesion that represents both a diagnostic and therapeutic dilemma. We report two cases of granulomatous mastitis recently evaluated and managed at our institution. To better understand this rare disease, we analyzed treatment outcomes in reported cases of granulomatous mastitis. One hundred sixteen cases were subsequently analyzed. Primary management strategies included observation (n = 9), steroids (n = 29), partial mastectomy (n = 75), and mastectomy (n = 3). Success rates with each treatment were observation, 56 per cent; steroids, 42 per cent; partial mastectomy, 79 per cent; and mastectomy, 100 per cent. Based on this analysis, we propose a clinically useful algorithm for both workup and management of these challenging cases.
The American surgeon 09/2007; 73(8):798-802. · 0.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: Histopathologic distinction between ductal and lobular carcinomas of the breast has been made since 1941. Together, these two subtypes account for >95% of all mammary carcinomas. With the recent advances in molecular techniques, our understanding of the biology behind these carcinomas has greatly expanded. The genomic aberrations in mammary carcinoma are highly complex and appear to be more associated with tumor grade rather than any histopathologic subtype. Protein and RNA expression profiling reveals a classification of mammary carcinoma that has some overlap with traditional histopathology and can at least partially explain clinical behavior. The goal of this review is to present what is currently known about the molecular profiles of infiltrating ductal and lobular carcinoma and how they relate to conventional histopathology and biologic behavior.
The Breast Journal 01/2007; 13(2):172-9. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Guidelines on thyroid fine-needle aspiration biopsy (FNABs) reporting calls for unambiguous diagnostic terminology in order to maximize treatment. This study evaluates how pathologists follow the guidelines and clinicians understand the diagnostic categories in terms of patient care.
Survey 1 asked pathologists who perform/interpret FNABs which of "atypical," "indeterminate," "suspicious," and "nondiagnostic" they routinely use. Survey 2 asked clinicians who treat thyroid nodules to correlate these categories to the options of "negative FNAB/follow-up," "repeat FNAB," and "proceed to surgery." The anonymous, voluntary results were entered into a database and analyzed.
Pathologists' and clinicians' response rates were 70% and 35%, respectively. Survey 1: 27% of pathologists used three, 27% used one, and 44% used two categories. Survey 2: 98% clinicians would repeat the FNAB with a "nondiagnostic" and 96% opted for surgery with a "suspicious" diagnosis. "Indeterminate" prompted 58% to repeat the FNAB and 32% to send the patient to surgery. "Atypical" would lead 37% to repeat the FNAB and 52% to send the patient to surgery.
Pathologists actively use the terminology "suspicious," "indeterminate," or "atypical," which cause confusion in some clinicians. These results support the need for a more standardized terminology for FNAB reporting and education of the clinicians on that terminology.
Thyroid: official journal of the American Thyroid Association 11/2006; 16(10):1003-8. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Approximately 70-80% of thyroid fine needle aspiration biopsies (FNAB) can distinguish benign from malignant thyroid nodules. However, much interpretive diagnostic difficulty arises with the remaining 20-30% of cases. These problematic thyroid aspirations have been placed in various diagnostic categories, which collectively have led to confusion and a negative impact on the clinical management of patients with thyroid nodules. We present our experience using a five-tier system, including the diagnostic terminology: benign, indeterminate, suspicious, malignant, and unsatisfactory.
Thyroid FNABs were diagnosed using a five-tier system from 200 consecutive patients and the subsequent surgical excisions were correlated.
Overall, there was an excellent association between the five diagnostic categories and predicting benign versus neoplastic thyroid nodules (LR = 96.06, X(2) = 76.49, and phi = 0.618, df = 4, p < 0.0001). A negative cytologic diagnosis carries a negative predictive value of 92%, while an indeterminate, suspicious, and malignant cytologic diagnosis carries a positive predictive value of 50, 71, and 100% respectively. The estimated sensitivity for an indeterminate, suspicious, or malignant cytologic diagnosis varied from 70 to 89%, while the specificity increased from 57 to 92 to 100%, respectively.
The data presented shows that the five diagnostic categories of thyroid FNAB are excellent at distinguishing benign from neoplastic thyroid nodules. Both the indeterminate and suspicious categories, while not statistically different from each other in predicting benign from neoplasia, are statistically different from obviously benign and obviously malignant categories. These results support the need for an indeterminate and/or suspicious category.
Thyroid: official journal of the American Thyroid Association 08/2006; 16(8):781-6. · 2.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fine-needle aspiration biopsy (FNAB) of thyroid nodules is a safe, cost-effective procedure but the rates of inadequate cytology specimens range from approximately 1% to 15%. This study tests the hypothesis that ultrasonographically (US) guided FNAB and onsite assessment of cytology improves the adequacy rate of FNAB. A retrospective analysis was performed on 693 thyroid FNAB specimens obtained with and without ultrasound guidance and with or without onsite cytology assessment. Overall, 29 specimens (4%) were inadequate for diagnosis. Among 163 cystic nodules and 530 solid nodules, inadequacy rates were 15% (n = 24) and 1% (n = 5) respectively (p = 0.0001). An onsite assessment of cytology for adequacy was done in 550 cases (83%), which was more accurately performed by a cytopathologist (97%) than a cytotechnologist (93%, p = 0.015). With US-guided FNAB, 3% of the cytology specimens were inadequate, compared to a 7% rate when US was not done (p = 0.003). The mean number of needle punctures necessary for an adequate specimen was 3.8 +/- 0.06 (median, 3.0; range, 1-11), which was different among various types of doctors, ranging from 3.2 +/- 0.07 to 5.4 +/- 0.12 (p = 0.001 analysis of variance [ANOVA]). The fewest number of needle passes to achieve an adequate specimen were required by university endocrinologists and pathologists working together (average, 3.2 +/- 0.07; median, 3.0; range, 1-11). Sample inadequacy rate varied significantly among physician groups, ranging from 3% to 18% (p = 0.0001 ANOVA). Stepwise regression analysis showed that onsite assessment of cytology, US-guided FNAB (p = 0.16), and cystic nature of the nodule (p < 0.0001 for all) correlated with adequacy of the specimen. We conclude that US-guided FNAB with onsite evaluation of cytology specimens substantially increases the adequacy of cytology specimens and decreases the number of required needle passes, which ultimately reduces patient discomfort and diagnostic errors, thus raising the question as to whether this should eventually become the standard of care. We believe this is a goal that training programs should strive to achieve.
[show abstract][hide abstract] ABSTRACT: Adenomyoma is a benign tumor composed of smooth muscle and benign endometrium. These tumors typically originate within the uterus. An extrauterine adenomyoma is a rare entity. We report a uterine-like mass consistent with an extrauterine adenomyoma presenting 22 years following a total abdominal hysterectomy and bilateral salpingo-oophorectomy. The mass was pear-shaped with uterine-type smooth muscle and a cavity lined by functional endometrial glands and stroma. To our knowledge, only 4 other cases of an extrauterine uterine-like mass are reported in the literature. Three involved the ovary, while one was located adjacent to the broad ligament with normal pelvic organs. Although none of these other uterus-like masses were described as adenomyomas with uterine-like features, the histologic findings are strikingly similar. An understanding of the müllerian system suggests that either an embryologic malformation or a differential multipotentiality existing in the subcoelomic tissues in response to hormonal stimulation results in a supernumerary müllerian structure like a uterus, as observed in this case. The presence of endometrial glands and stroma in the mass confirms that the tissues in this mass are hormonally responsive. It is most likely that this uterine-like mass arose from the tissues of the secondary müllerian system in response to estrogenic stimulation.
Archives of pathology & laboratory medicine 09/2005; 129(8):1041-3. · 2.78 Impact Factor
[show abstract][hide abstract] ABSTRACT: Does papillary squamotransitional cell carcinoma (PSTCC) behave differently from conventional squamous cell carcinoma of the cervix and does PSTCC have true transitional cell differentiation?
Twenty cases of PSTCC were identified from archival files. Clinical data were compiled. Immunoperoxidase stains for uroplakin III, p63 and p16 were performed on available tissue blocks.
Patients ranged in age from 27 to 85 years. Twelve patients were FIGO Stage I, 4 were Stage II, and 2 were Stage III. Thirteen patients had clinical follow-up ranging from 5 to 132 months. Three patients subsequently had more extensive disease than initial clinical staging indicated. Nine patients had no tumor progression, three had local recurrence and one had metastatic disease. Eight cases were strongly immunoreactive for p63 and p16 and 14 were negative for uroplakin III.
PSTCC lacks true transitional cell differentiation and probably shares similar clinicopathologic features with conventional cervical squamous cell carcinoma.
[show abstract][hide abstract] ABSTRACT: Vulvar intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. The purpose of this study was to investigate the functional properties of VIN related to expression of p16(INK4a) protein as well as to detection of human papillomavirus (HPV) type 16 by real-time polymerase chain reaction (RT-PCR) analysis.
A total of 49 vulvar biopsy samples were examined by hematoxylin-eosin staining from benign/reactive lesions, condyloma acuminatum, VIN, and invasive squamous cell carcinoma (SCC). JC8 mouse monoclonal antibodies were used that recognize p16(INK4a) epitope at a dilution of 1:25. The reaction pattern for p16(INK4a) was graded in each sample between 0 and 3+. RT-PCR analysis of formalin-fixed paraffin-embedded sections determined positivity for HPV type 16.
p16(INK4a) immunoreactivity was different in VIN 1, VIN 2, VIN 3, and squamous cell carcinoma. Strong expression of p16(INK4a) protein was observed in 92% (22 of 24) of VIN 2 and VIN 3 lesions and 100% (4 of 4) of invasive SCCs. Two (67%) of 3 VIN 2 lesions, 17 (81%) of 21 VIN 3 lesions, and 4 (100%) of 4 SCCs were positive for HPV type 16 by PCR analysis. Two (20%) of 10 VIN 1 lesions were immunoreactive for p16(INK4a), with only 1 lesion positive for HPV type 16. No p16(INK4a) immunoreactivity was observed in any of the benign/reactive and condyloma acuminatum lesions. In addition, none of the benign/reactive or condyloma lesions were positive for HPV type 16 by RT-PCR analysis.
Upregulation of INK4a gene occurs in vulvar carcinogenesis. p16(INK4a) is not a sensitive marker for differentiation of benign vulvar squamous epithelium from condyloma acuminatum or VIN 1 lesions because most VIN 1 lesions are p16(INK4a) negative. Expression of p16(INK4a) may aid in the diagnosis of HPV-related lesions and as such may be of value as a surrogate marker in the diagnosis of vulvar premalignant and malignant lesions.