Inhibition of the integrin/FAK signaling axis and c-Myc synergistically disrupts ovarian cancer malignancy.

Integrins, a family of heterodimeric receptors for extracellular matrix, are promising therapeutic targets for ovarian cancer, particularly high-grade serous-type (HGSOC), as they drive tumor cell attachment, migration, proliferation and survival by activating focal adhesion kinase (FAK)-dependent signaling. Owing to the potential off-target effects of FAK inhibitors, disruption of the integrin signaling axis remains to be a challenge. Here, we tackled this barrier by screening for inhibitors being functionally cooperative with small-molecule VS-6063, a phase II FAK inhibitor. From this screening, JQ1, a potent inhibitor of Myc oncogenic network, emerged as the most robust collaborator. Treatment with a combination of VS-6063 and JQ1 synergistically caused an arrest of tumor cells at the G2/M phase and a decrease in the XIAP-linked cell survival. Our subsequent mechanistic analyses indicate that this functional cooperation was strongly associated with the concomitant disruption of activation or expression of FAK and c-Myc as well as their downstream signaling through the PI3K/Akt pathway. In line with these observations, we detected a strong co-amplification or upregulation at genomic or protein level for FAK and c-Myc in a large portion of primary tumors in the TCGA or a local HGSOC patient cohort. Taken together, our results suggest that the integrin-FAK signaling axis and c-Myc synergistically drive cell proliferation, survival and oncogenic potential in HGSOC. As such, our study provides key genetic, functional and signaling bases for the small-molecule-based co-targeting of these two distinct oncogenic drivers as a new line of targeted therapy against human ovarian cancer.

SUSD2 expression in high-grade serous ovarian cancer correlates with increased patient survival and defective mesothelial clearance.

The cause of death among the majority of epithelial ovarian cancer (EOC) patients involves passive dissemination of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids into adjacent organs. Thus, it is important to identify the factors that mediate EOC metastasis and implantation, including clearance of the mesothelium. Sushi domain containing 2 (SUSD2) encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. Immunohistochemical analysis determined the presence of SUSD2 in several subtypes of EOC, with the strongest staining observed in high-grade serous ovarian carcinomas (HGSOCs). A high-density, clinically annotated HGSOC tissue microarray was stained with an anti-SUSD2 antibody. Patients with tumors that had a low percentage of SUSD2 staining cells had a shorter median survival (31.7 months) compared with patients who had tumors with extensive SUSD2 staining (49.1 months; P-value=0.0083). To investigate the role of SUSD2 in HGSOCs, stable OVCAR3, OVSAHO and KURAMOCHI cell lines were established with knockdown (KD) or non-targeting (NT) of SUSD2. Boyden chamber and wound-healing assays demonstrated that OVCAR3, OVSAHO and KURAMOCHI SUSD2-KD cells migrated at significantly higher rates compared with their SUSD2 NT counterpart cell lines. Quantitative reverse transcription-PCR and western immunoblot analysis indicated an inverse relationship between SUSD2 and well-characterized mesenchymal proteins, including Twist-1, Zeb-1, N-cadherin, STEAP1, AHNAK, Snail-1, COL5A2 and Snail-3 in OVCAR3, OVSAHO and KURAMOCHI cell line models. In addition, OVCAR3 and KURAMOCHI SUSD2-KD spheroids displayed increased mesothelial clearance ability compared with cells that express endogenous levels of SUSD2. These data suggest that SUSD2 has a role in the inhibition of mesothelial clearance, which is required for metastasis. Altogether, our findings indicate that SUSD2 impedes migration, epithelial-to-mesenchymal transitional and mesothelial clearance of HGSOC cells, consistent with prolonged survival of patients with SUSD2-expressing tumors.

YAP induces high-grade serous carcinoma in fallopian tube secretory epithelial cells.

Accumulating evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from fallopian tube secretory epithelial cells (FTSECs). However, the molecular mechanisms underlying the initiation and progression of HGSC derived from FTSECs remains unclear. In this study, we found that the Hippo/Yes-associated protein (YAP) signaling pathway has a critical role in the initiation and progression of fallopian tube and ovarian HGSC. Importantly, YAP was overexpressed in inflammatory and cancerous fallopian tube tissues. Further, overexpression of wild-type YAP, or constitutively active YAP in immortalized FTSECs, induced cell proliferation, migration, colony formation and tumorigenesis. Moreover, the Hippo/YAP and the fibroblast growth factor (FGF) signaling pathways formed an autocrine/paracrine-positive feedback loop to drive the progression of the FTSEC-derived HGSC. Evidence in this study strongly suggests that combined therapy with inhibitors of YAP (such as verteporfin) and FGF receptors (such as BGJ398) can provide a novel therapeutic strategy to treat fallopian tube and ovarian HGSC.

Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors.

High-grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirm that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through the activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into the examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.

FOXO3a loss is a frequent early event in high-grade pelvic serous carcinogenesis.

Serous ovarian carcinoma is the most lethal gynecological malignancy in Western countries. The molecular events that underlie the development of the disease have been elusive for many years. The recent identification of the fallopian tube secretory epithelial cells (FTSECs) as the cell-of-origin for most cases of this disease has led to studies aimed at elucidating new candidate therapeutic pathways through profiling of normal FTSECs and serous carcinomas. Here we describe the results of transcriptional profiles that identify the loss of the tumor suppressive transcription factor FOXO3a in a vast majority of high-grade serous ovarian carcinomas. We show that FOXO3a loss is a hallmark of the earliest stages of serous carcinogenesis and occurs both at the DNA, RNA and protein levels. We describe several mechanisms responsible for FOXO3a inactivity, including chromosomal deletion (chromosome 6q21), upregulation of miRNA-182 and destabilization by activated PI3K and MEK. The identification of pathways involved in the pathogenesis of ovarian cancer can advance the management of this disease from being dependant on surgery and cytotoxic chemotherapy alone to the era of targeted therapy. Our data strongly suggest FOXO3a as a possible target for clinical intervention.

Coming into focus: the nonovarian origins of ovarian cancer.

BACKGROUND: The traditional view of epithelial ovarian cancer asserts that all tumor subtypes share a common origin in the ovarian surface epithelium (OSE) DESIGN: A literature review was carried out to summarize the emerging understanding of extraovarian sources of epithelial ovarian carcinomas. RESULTS: Historically, there were no diagnostic criteria for documenting the origin of ovarian epithelial carcinomas. Moreover, there are no normal epithelial tissues in the ovary with morphologic similarities to these tumors. In fact, no precursor lesions have ever been reproducibly identified in the ovary. However, there is a strong correlation between extrauterine Müllerian tissue and the development of ovarian carcinomas, tumors of low malignant potential, and cystadenomas. The most recent support for this hypothesis comes from the careful analysis of risk-reducing bilateral salpingo-oopherectomy specimens from BRCA1 or BRCA2 mutation carriers. These studies showed that a significant majority of high-grade serous ovarian carcinomas, the most common subtype, arise from the fallopian tube fimbriae rather than the OSE. CONCLUSIONS: Mounting evidence indicates that the vast majority of epithelial ovarian carcinomas are not ovarian in origin. Extrauterine Müllerian epithelium from various sites in the reproductive tract likely accounts for the diverse morphology and behavior of these tumors.

Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis.

Recent studies suggest that some serous ovarian carcinomas (SOCs) arise from the fallopian tube (FT) epithelium rather than the ovarian surface epithelium. This hypothesis places emphasis on the FT secretory epithelial cell as a cell-of-origin. Herein, we report the development of a novel ex vivo primary human FT epithelium culture system that faithfully recapitulates the in vivo epithelium, as shown by morphological, ultrastructural and immunophenotypic analyses. Mass spectrometry-based proteomics reveal that these cultures secrete proteins previously identified as biomarkers for ovarian cancer. We also use this culture system to study the response of the FT epithelium to genotoxic stress and find that the secretory cells exhibit a distinct response to DNA damage when compared with neighboring ciliated cells. The secretory cells show a limited ability to resolve the damage over time, potentially leaving them more susceptible to accumulation of additional mutagenic injury. This divergent response is confirmed with in situ studies using tissue samples, further supporting the use of this ex vivo culture system to investigate FT epithelial pathobiology. We anticipate that this novel culture system will facilitate the study of SOC pathogenesis, and propose that similar culture systems could be developed for other organ site-specific epithelia.

Tubal and ovarian pathways to pelvic epithelial cancer: a pathological perspective.

Prolongation of ovarian epithelial cancer survival depends on early detection or improved responses to chemotherapy. Gains in either have been modest at best. Understanding the diverse pathogenesis of this disease is critical to early intervention or prevention. This review addresses six important variables, including (i) cell of origin, (ii) site of origin, (iii) initial genotoxic events, (iv) risks imposed by hereditary and other promoting conditions, (v) subsequent factors that promote different patterns of metastatic spread, and (vi) prospects for intervention. This review proposes two distinct pathways to pelvic epithelial cancer. The first initiates in ovarian surface epithelium (OSE), Mullerian inclusions or endometriosis in the ovary. The second arises from the endosalpinx and encompasses a subset of serous carcinomas. The serous carcinogenic sequence in the distal fallopian tube is described and contrasted with lower grade serous tumors based on tumour location, earliest genetic change and ability (or lack of) to undergo terminal (ciliated) differentiation. Ultimately, a clear understanding of tumour origin and the mechanism(s) leading to the earliest phases of the serous and endometrioid carcinogenic sequences may hold the greatest promise for designing prevention strategies and/or developing new therapies.

A candidate precursor to serous carcinoma that originates in the distal fallopian tube.

The tubal fimbria is a common site of origin for early (tubal intraepithelial carcinoma or TIC) serous carcinomas in women with familial BRCA1 or 2 mutations (BRCA+). Somatic p53 tumour suppressor gene mutations in these tumours suggest a pathogenesis involving DNA damage, p53 mutation, and progressive loss of cell cycle control. We recently identified foci of strong p53 immunostaining-termed 'p53 signatures'-in benign tubal mucosa from BRCA+ women. To examine the relationship between p53 signatures and TIC, we compared location (fimbria vs ampulla), cell type (ciliated vs secretory), evidence of DNA damage, and p53 mutation status between the two entities. p53 signatures were equally common in non-neoplastic tubes from BRCA+ women and controls, but more frequently present (53%) and multifocal (67%) in fallopian tubes also containing TIC. Like prior studies of TIC, p53 signatures predominated in the fimbriae (80-100%) and targeted secretory cells (HMFG2 + /p73-), with evidence of DNA damage by co-localization of gamma-H2AX. Laser-capture microdissected and polymerase chain reaction-amplified DNA revealed reproducible p53 mutations in eight of 14 fully-analysed p53 signatures and all of the 12 TICs; TICs and their associated ovarian carcinomas shared identical mutations. In one case, a contiguous p53 signature and TIC shared the same mutation. Morphological intermediates between the two, with p53 mutations and moderate proliferative activity, were also seen. This is the first report of an early and distinct alteration in non-neoplastic upper genital tract mucosa that fulfils many requirements for a precursor to pelvic serous cancer. The p53 signature and its malignant counterpart (TIC) underline the significance of the fimbria, both as a candidate site for serous carcinogenesis and as a target for future research on the early detection and prevention of this disease.

The p400 complex is an essential E1A transformation target.

Here, we report the identification of a new E1A binding protein complex that is essential for E1A-mediated transformation. Its core component is a SWI2/SNF2-related, 400 kDa protein (p400). Other components include the myc- and p/CAF-associated cofactor, TRRAP/PAF400, the DNA helicases TAP54alpha/beta, actin-like proteins, and the human homolog of the Drosophila Enhancer of Polycomb protein. An E1A mutant, defective in p400 binding, is also defective in transformation. Certain p400 fragments partially rescued this phenotype, underscoring the role of E1A-p400 complex formation in the E1A transforming process. Furthermore, E1A and c-myc each alter the subunit composition of p400 complexes, implying that physiological p400 complex formation contributes to transformation suppression.

BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function.

BRCA1 interacts in vivo with a novel protein, BACH1, a member of the DEAH helicase family. BACH1 binds directly to the BRCT repeats of BRCA1. A BACH1 derivative, bearing a mutation in a residue that was essential for catalytic function in other helicases, interfered with normal double-strand break repair in a manner that was dependent on its BRCA1 binding function. Thus, BACH1/BRCA1 complex formation contributes to a key BRCA1 activity. In addition, germline BACH1 mutations affecting the helicase domain were detected in two early-onset breast cancer patients and not in 200 matched controls. Thus, it is conceivable that, like BRCA1, BACH1 is a target of germline cancer-inducing mutations.

Pentostatin and rituximab in the treatment of patients with B-cell malignancies.

Both pentostatin (Nipent) and rituximab (Rituxan) have single-agent activity in B-cell malignancies, including indolent and intermediate-grade non-Hodgkin's lymphoma (NHL). Pentostatin is also active in pretreated patients with chronic lymphocytic leukemia (CLL). In spite of current treatment modalities, few patients with these diseases are cured. The combination of rituximab and pentostatin is an attractive treatment option because both drugs have a limited toxicity profile and can be delivered on an outpatient basis. We describe the design of a phase II multicenter study to evaluate the safety and efficacy of pentostatin in combination with rituximab in patients with previously treated and untreated low-grade NHL and CLL.

Infectious complications of pentostatin therapy.

Managing the infectious complications associated with pentostatin (Nipent), used alone or in combination with other agents in patients with low-grade lymphomas, poses a significant problem for clinicians. Since there is limited experience with these therapies, definitive treatment recommendations concerning prophylaxis cannot be made. The panel members discussed the use of valacyclovir (Valtrex) to provide prophylaxis for herpes zoster, trimethoprim/sulfamethoxazole for Pneumocystis, and acyclovir (Zovirax) for varicella zoster. They also considered combinations of pentostatin with agents such as interferon, rituximab (Rituxan), and chlorambucil (Leukeran) and their effect on the immune system. The biology of B and T cells was discussed, with an emphasis on clinical application.

Effect of granisetron on performance status during high-dose interferon therapy.

Two case reports demonstrating the new complete amelioration of the toxicity of alpha-2b recombinant interferon using granisetron i.v. or p.o.

Immunoaffinity purification of the human multisubunit transcription factor IIH.

A procedure to immunoaffinity purify the human transcription factor IIH (TFIIH) was developed using a monoclonal antibody that recognizes an epitope in ERCC3 (XPB), the largest subunit of TFIIH. The epitope recognized by the monoclonal antibody was mapped to 20 amino acids. A peptide containing the epitope was capable of displacing TFIIH from an immunoaffinity column containing the monoclonal antibody. The immunoaffinity purification procedure described allows a simple and efficient method to purify both the "core" and "holo" TFIIH complexes.

Engraftment and outcomes of patients receiving myeloablative therapy followed by autologous peripheral blood stem cells with a low CD34+ cell content.

Engraftment kinetics after high-dose chemotherapy (HDC) were evaluated in patients receiving autologous peripheral blood stem cell (PBSC) infusions with a low CD34+ cell content. Forty-eight patients were infused with < 2.5 x 10(6) CD34+ cells/kg; 36 because of poor harvests and 12 because they electively received only a fraction of their harvested cells. A median of 2.12 x 10(6) CD34+ cells/kg (range, 1.17-2.48) were infused following one of seven different HDC regimens. All patients achieved absolute neutrophil counts > or = 0.5 x 10(9)/l at a median of day 11 (range, 9-16). Forty-seven patients achieved platelet counts > or = 20 x 10(9)/l at a median of day 14 (range, 8-250). Nine of 47 (19%) had platelet recovery after day 21, 4/47 (9%) after day 100 and one died on day 240 without platelet recovery. Twenty-six patients (54%) died of progressive disease in 51-762 days; 22 (46%) are alive at a median of 450 days (range, 94-1844), 17 (35%) of whom are surviving disease-free at a median of 494 days (range, 55-1263). No patient died as a direct consequence of low blood cell counts. These data demonstrate that PBSC products containing 1.17-2.48 x 10(6) CD34+ cells/kg resulted in relatively prompt neutrophil recovery in all patients but approximately 10% had delayed platelet recovery.

Collection of peripheral blood stem cells following administration of paclitaxel, cyclophosphamide, and filgrastim in patients with breast and ovarian cancer.

PURPOSE: To determine the toxicities and efficacy of paclitaxel, cyclophosphamide (Cy), and recombinant human granulocyte-colony stimulating factor (filgrastim) administered for mobilization and collection of peripheral blood stem cells (PBSC) in patients with breast and ovarian cancer. METHODS: One hundred and forty-one patients with breast (n = 115) or ovarian cancer (n = 26) received paclitaxel 170 mg/m2 and Cy 2 gm/m2 (n = 42) or paclitaxel 200 mg/m2, Cy 3 gm/m2 (n = 99), and filgrastim (6 micrograms/kg/day) followed by collection of PBSC by apheresis. RESULTS: The 2 dose levels of paclitaxel and Cy tested were well tolerated. The median yield of CD34+ cells from all patients was 6.53 x 10(6)/kg (range, 0.11-51.76) collected with a median of 2 aphereses (range, 1-8). The target dose of 2.5 x 10(6) CD34+ cells/kg was achieved in 85% of patients. The mean daily collection of CD34+ cells was 5.46 x 10(6)/kg for patients receiving 200 mg/m2 of paclitaxel and 3 gm/m2 of Cy as compared to 2.77 for patients receiving the lower doses (p = 0.0005). Increasing the dose of paclitaxel and Cy did not significantly increase the fraction of patients achieving a target dose of 2.5 x 10(6) CD34+ cells/kg (87% vs 81%, p = 0.367) but did increase the fraction achieving a target of 5.0 x 10(6) CD34+ cells/kg (73% vs 45%, p = 0.002). The mean daily collection of CD34+ cells for patients who had received only 1 prior chemotherapy regimen was 6.59 x 10(6)/kg as compared to 3.47 for patients who had received more than 1 prior chemotherapy regimen (p < 0.0001). Prior radiation therapy (p = 0.003) and patient performance status (p = 0.047) were adverse risk factors for achieving a target dose of > or = 2.5 x 10(6) CD34+ cells/kg. CONCLUSIONS: The combination of paclitaxel, Cy, and filgrastim can be administered with acceptable toxicity, allowing collection of adequate quantities of PBSC from the majority of patients with breast and ovarian cancer. Increasing the doses of paclitaxel and Cy increased the number of CD34+ cells collected and decreased the number of apheresis procedures necessary to collect target cell doses. However, increasing drug doses did not increase the fraction of patients yielding the minimum CD34+ target dose of 2.5 x 10(6)/kg. Collection of PBSC early in the disease course is the best strategy to assure optimal CD34+ cell doses in all patients.

RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair.

Bulky lesions in the template strand block the progression of RNA polymerase II (RNAP II) and are repaired more rapidly than lesions in the non-transcribed strand, which do not block transcription. In order to better understand the basis of this transcription-coupled repair we developed an in vitro system with purified transcription and nucleotide excision repair proteins and a plasmid containing the adenovirus major late promoter and a thymine dimer in the template strand downstream of the transcription start site. The footprint of RNAP II stalled at the thymine dimer, obtained using DNase I, lambda exonuclease and T4 polymerase 3'-->5'exonuclease, covers approximately 40 nt and is nearly symmetrical around the dimer. The ternary complex formed at the lesion site is rather stable, with a half-life of approximately 20 h. Surprisingly, addition of human repair proteins results in repair of transcription-blocking dimers in the ternary complex. The blocked polymerase neither inhibits nor stimulates repair and repair is observed in the absence of CSB protein, the putative human transcription-repair coupling factor.

Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.

Transcription factor IIH (TFIIH) is a multisubunit complex required for transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits.