Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness

Paul P. Szotek^*, Rafael Pieretti-Vanmarcke^*, Peter T. Masiakos^*, Daniela M. Dinulescu, Denise Connolly, Rosemary Foster, David Dombkowski^¶, Frederic Preffer^¶, David T. MacLaughlin^*, and Patricia K. Donahoe^*,||

*Pediatric Surgical Research Laboratories, Department of Surgery, and ^¶Flow Cytometry Laboratory, Department of Pathology and Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114; Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111; Department of Pathology, Eugene Braunwald Research Center, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Avenue, Room 401a, Boston, MA 02115; and Department of Medicine, Division of Hematology/Oncology, Massachusetts General Hospital, Harvard Medical School, 70 Blossom Street, Boston, MA 02114

The recent identification of “side population” (SP) cells in a number of unrelated human cancers and their normal tissue sources has renewed interest in the hypothesis that cancers may arise from somatic stem/progenitor cells. The high incidence of recurrence attributable to multidrug resistance and the multiple histologic phenotypes indicative of multipotency suggests a stem cell-like etiology of ovarian cancer. Here we identify and characterize SP cells from two distinct genetically engineered mouse ovarian cancer cell lines. Differential efflux of the DNA-binding dye Hoechst 33342 from these cell lines defined a human breast cancer-resistance protein 1-expressing, verapamil-sensitive SP of candidate cancer stem cells. In vivo, mouse SP cells formed measurable tumors sooner than non-SP (NSP) cells when equal numbers were injected into the dorsal fat pad of nude mice. The presence of Mullerian Inhibiting Substance (MIS) signaling pathway transduction molecules in both SP and NSP mouse cells led us to investigate the efficacy of MIS against these populations in comparison with traditional chemotherapies. MIS inhibited the proliferation of both SP and NSP cells, whereas the lipophilic chemotherapeutic agent doxorubicin more significantly inhibited the NSP cells. Finally, we identified breast cancer-resistance protein 1-expressing verapamil-sensitive SPs in three of four human ovarian cancer cell lines and four of six patient primary ascites cells. In the future, individualized therapy must incorporate analysis of the stem cell-like subpopulation of ovarian cancer cells when designing therapeutic strategies for ovarian cancer patients.

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Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration.

Methods

Human ovarian carcinoma cell line, ES-2 cells were treated with various concentrations of TGZ. Cell migration was evaluated by wound-healing and Boyden chamber transwell experiments. PPAR gamma expression was blocked by PPAR gamma small interfering RNA. The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies. The cellular distribution of paxillin, vinculin, stress fiber and PTEN was assessed by immunocytochemistry.

Results

TGZ dose- and time-dependently impaired cell migration through a PPAR gamma independent manner. TGZ treatment impaired cell spreading, stress fiber formation, tyrosine phosphorylation of focal adhesion kinase (FAK), and focal adhesion assembly in cells grown on fibronectin substratum. TGZ also dose- and time-dependently suppressed FAK autophosphorylation and phosphorylation of the C-terminal of PTEN (a phosphatase). At concentration higher than 10 micromolar, TGZ caused accumulation of PTEN in plasma membrane, a sign of PTEN activation.

Conclusion

These results indicate that TGZ can suppress cultured ES-2 cells migration. Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity.

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A new study published in CANCER found that cigarette smoking and alcohol consumption do increase ovarian cancer risk while caffeine may lower the risk, particularly in women not using hormones.

Various studies have assessed the potential link between modifiable factors such as smoking or caffeine and alcohol intake and have generated conflicting results. To help clarify these associations, Dr. Shelley S. Tworoger, of Harvard Medical School and the Harvard School of Public Health, and colleagues examined ongoing questionnaire data from the Brigham and Women’s Hospital-based Nurses’ Health Study, which includes 121,701 US female registered nurses. The Nurses’ Health Study cohort was established in 1976, when women aged 30-35 completed and returned initial questionnaires. Every two years, questionnaires are sent to the women to update exposure variables and document newly diagnosed diseases.

Dr. Tworoger and her co-investigators prospectively examined associations between smoking and ovarian cancer risk among 110,454 women and between alcohol or caffeine and ovarian cancer risk among 80,253 women, all followed between June 1, 1976 and June 1, 2004. For the smoking analyses, they identified 737 confirmed cases of epithelial ovarian cancer, and for the dietary analyses, they identified 507 cases.

There was no association between current or past smoking and ovarian cancer risk, however smoking status, duration, and pack-years were significantly associated with risk of mucinous tumors, a rare form of ovarian cancer. The authors also found no association between alcohol consumption and ovarian cancer risk. However they observed an inverse trend of risk with total caffeine and caffeinated coffee intake, but no association with decaffeinated coffee. The potential reduction in risk with higher caffeine intake appeared to be strongest for women who had never used oral contraceptives or postmenopausal hormones.

The authors concluded that “reducing alcohol intake and cessation of smoking is not likely to have a substantial impact on risk of ovarian cancer.” They add that “the possibility that caffeine may reduce ovarian cancer risk, particularly for women who have not previously used exogenous hormones, is intriguing and warrants further study, including an evaluation of possible biological mechanisms.”

Article: “Caffeine, Alcohol, Smoking, and the Risk of Incident Epithelial Ovarian Cancer,” Shelley S. Tworoger, Dorota M. Gertig, Margaret A. Gates, Jonathan L. Hecht, and Susan E. Hankinson. CANCER;DOI: 10.1002/cncr.23275

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Advocates and researchers at the University of Michigan hope that increased awareness about ovarian cancer can save lives.

Here is what they want you to know.

Symptoms do exist. Bloating, pelvic or abdominal pain, difficulty eating, feeling full quickly, and frequent or urgent urinating are have been shown to be related to a possible case of ovarian cancer and if they persist you should see your doctor.

1.        There is no early detection screening test currently.

2.        All women should get yearly pelvic exams.

3.        Ovarian cancer is difficult to treat because it’s often resistant to current treatments.

4.        Survival rates are better at the early stage.

5.        Most common in older white women.

6.        A small number of ovarian cancers are hereditary, linked to the same genes that are linked to breast cancer, BRCA1 and BRCA2.

7.        The best person to treat ovarian cancer is a gynecologic oncologist.

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Ko-Hui Tung1, Marc T. Goodman1 , Anna H. Wu2, Katharine McDuffie1, Lynne R. Wilkens1, Laurence N. Kolonel1, Abraham M. Y. Nomura1, Keith Y. Terada3, Michael E. Carney3 and Leslie H. Sobin4

1 Cancer Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI.
2 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA.
3 Department of Obstetrics and Gynecology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI.
4 World Health Organization Collaborating Center for International Histological Classification of Tumors, Armed Forces Institute of Pathology, Washington, DC.

Associations of reproductive factors with ovarian cancer may differ by histologic type. Data from a multiethnic, population-based, case-control study, conducted in Hawaii and Los Angeles, California, between 1993 and 1999, were used to assess this hypothesis. A structured questionnaire was administered to 558 histologically confirmed epithelial ovarian cancer cases and 607 population controls. Factors suppressing ovulation, including pregnancy and oral contraceptive use, were inversely associated with the risk of all histologic types. Nonmucinous but not mucinous tumors were significantly associated with menstruation years (odds ratio = 1.5 for the highest vs. the lowest quartile) and lifetime ovulatory cycles (odds ratio = 2.8 for the highest vs. the lowest quartile). Duration of breastfeeding (odds ratio = 0.4 for the highest vs. the lowest quartile) was significantly and inversely related to nonmucinous tumors but not to mucinous tumors. Among all tumor types, endometrioid tumors were the most strongly related to pregnancy and tubal ligation, while clear cell tumors were the only type that was associated with noncontraceptive hormone use. The risk factors were similar for borderline and invasive tumors, except for age at diagnosis. Mucinous tumors, both borderline and invasive, were more common in Asian women than in Caucasian and other women. Our data suggest that histologic types of epithelial ovarian cancer are etiologically distinct.

Breast feeding; case-control studies; contraceptives, oral; histology; ovarian neoplasms; ovulation; parity; pregnancy

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One reason that ovarian cancer is such a deadly disease is because it is not usually diagnosed until it has reached an advanced stage. In this study, we developed a novel algorithm for group biomarkers identification using gene expression data. Group biomarkers consist of coregulated genes across normal and different stage diseased tissues. Unlike prior sets of biomarkers identified by statistical methods, genes in group biomarkers are potentially involved in pathways related to different types of cancer development. They may serve as an alternative to the traditional single biomarkers or combination of biomarkers used for the diagnosis of early-stage and/or recurrent ovarian cancer. We extracted group biomarkers by applying biclustering algorithms that we recently developed on the gene expression data of over 400 normal, cancerous, and diseased tissues. We identified several groups of coregulated genes that encode for secreted proteins and exhibit expression levels in ovarian cancer that are at least 2-fold (in log₂ scale) higher than in normal ovary and nonovarian tissues. In particular, three candidate group biomarkers exhibited a conserved biological pattern that may be used for early detection or recurrence of ovarian cancer with specificity greater than 99% and sensitivity equal to 100%. We validated these group biomarkers using publicly available gene expression data sets downloaded from a NIH Web site. Statistical analysis showed that our methodology identified an optimum combination of genes that have the highest effect on the diagnosis of the disease compared with several computational techniques that we tested. Our study also suggests that single or group biomarkers correlate with the stage of the disease. [Mol Cancer Ther 2008;7(1):27–37]

Footnotes

Grant support: University of Minnesota Graduate Program Grant-in-Aid of Research, Artistry, and Scholarship Program and NIH/National Cancer Institute grant NIH R01CA106878 (A.P.N. Skubitz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Background

Ovarian cancer is a devastating disease partly because conventional chemotherapy ignores aspects of tumour biology. Recurrence represents the “true killer” among ovarian cancer patients and identification of novel molecular markers present in recurrent tumours is urgently required. We carried out gene expression profiling between primary and recurrent ovarian cancers and identified potential biomarkers of recurrence.

Results

Using cDNA microarrays, we identified distinct patterns of gene expression between primary and recurrent ovarian cancers from different patients with the same histology and from the same patients with different histology. Selected targets were validated and correlated with microarray results. Signatures from both cohorts were also validated against an independent set of serous papillary ovarian adenocarcinomas. Notably, upregulated genes in the recurrent compared to primary tumours in both cohorts, segregated in the same gene families.

Conclusions

Collectively, our data propose an integrative model for recurrence in ovarian cancer, in which tumour cells during relapse produce adhesion molecules to mediate attachment, cytokines and inflammatory mediators to stimulate survival and a variety of growth factors bound to their cognate receptors to fully proliferate in order to confront and modulate their immediate environment. Some of the mechanisms involved in recurrence could be specific to the drugs used.

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Symptoms

Ovarian cancer does not cause many symptoms in its early stages. This is why 75% to 80% of cases are not found until the cancer has spread. ² Most women do have symptoms in the 6 to 12 months before ovarian cancer is found. Symptoms that occur in later stages are most likely caused by the pressure of the growing cancer. These symptoms include:

—                  Ongoing cramps or pain in your belly.

—                  Ongoing pain in your pelvis or lower back.

—                  Abnormal bleeding from your vagina, especially after menopause if you are not using any hormonal medicines.

—                  Abnormal discharge from your vagina that contains mucus that may be tinged with blood.

—                  Pain or bleeding during sex.

—                  Nausea or loss of appetite or you cannot eat normally.

—                  Ongoing bloating or intestinal gas that is not relieved by home treatment measures.

—                  Bigger belly size or a lump that can be felt in your belly.

—                  Decreased energy level.

—                  A change in your bowel habits, such as constipation or diarrhea.

—                  A change in your bladder habits, such as urinary frequency or urgency.

—                  Weight loss.

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Background Standard chemotherapy for newly diagnosed ovarian cancer is a platinum–taxane combination. The Gynecologic Oncology Group conducted a randomized, phase 3 trial that compared intravenous paclitaxel plus cisplatin with intravenous paclitaxel plus intraperitoneal cisplatin and paclitaxel in patients with stage III ovarian cancer.

Methods We randomly assigned patients with stage III ovarian carcinoma or primary peritoneal carcinoma with no residual mass greater than 1.0 cm to receive 135 mg of intravenous paclitaxel per square meter of body-surface area over a 24-hour period followed by either 75 mg of intravenous cisplatin per square meter on day 2 (intravenous-therapy group) or 100 mg of intraperitoneal cisplatin per square meter on day 2 and 60 mg of intraperitoneal paclitaxel per square meter on day 8 (intraperitoneal-therapy group). Treatment was given every three weeks for six cycles. Quality of life was assessed.

Results Of 429 patients who underwent randomization, 415 were eligible. Grade 3 and 4 pain, fatigue, and hematologic, gastrointestinal, metabolic, and neurologic toxic effects were more common in the intraperitoneal-therapy group than in the intravenous-therapy group (P0.001). Only 42 percent of the patients in the intraperitoneal-therapy group completed six cycles of the assigned therapy, but the median duration of progression-free survival in the intravenous-therapy and intraperitoneal-therapy groups was 18.3 and 23.8 months, respectively (P=0.05 by the log-rank test). The median duration of overall survival in the intravenous-therapy and intraperitoneal-therapy groups was 49.7 and 65.6 months, respectively (P=0.03 by the log-rank test). Quality of life was significantly worse in the intraperitoneal-therapy group before cycle 4 and three to six weeks after treatment but not one year after treatment.

Conclusions As compared with intravenous paclitaxel plus cisplatin, intravenous paclitaxel plus intraperitoneal cisplatin and paclitaxel improves survival in patients with optimally debulked stage III ovarian cancer.

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Use of oral contraceptives during a woman’s life-time gives substantial long-term protection against ovarian cancer - and the longer they are used, the greater the reduction in risk. These are the conclusions of authors of an Article in this week’s edition of The Lancet.

Use of oral contraceptives has long been known to reduce the incidence of ovarian cancer. Because ovarian cancer is not common in young women and the incidence increases with age, the public-health effect of this reduction depends mainly on how the reduced risk persists decades after oral contraceptive use ceases.

Professor Valerie Beral and colleagues from the Collaborative Group on Epidemiological Studies of Ovarian Cancer have brought together 45 epidemiological studies of ovarian cancer to prepare the Article. They studied 23 257 women with ovarian cancer, 7308 (31 per cent) of whom had ever used oral contraceptives; and 87303 women without ovarian cancer, of which 32717 (37 per cent) had ever used oral contraceptives. In women with ovarian cancer, the mean age of diagnosis was 56 years and the median year of diagnosis was 1993. Those that had used contraceptives had done so for an average of 4.4 years in the ovarian cancer group and 5.0 years in the control group.

The researchers found that in high income countries, ten years use of oral contraceptives was estimated to reduce ovarian cancer incidence before age 75 from 12 per 1000 women to eight per 1000, and mortality from seven per 1000 women to five per 1000. For every 5000 woman-years of oral contraceptive use (eg, if 5000 women used oral contraceptives for one year), about two ovarian cancers and one death from the disease before age 75 are prevented.

Further, they found that although oestrogen doses in oral contraceptives have decreased significantly over the years (preparations in the 1960s typically contained more than double the oestrogen dose of preparations in the 1980s), there was no apparent variation in the relative risk of ovarian cancer between women whose oral contraceptive use was during the 1960s, 1970s or 1980s.

The risk reduction did not vary substantially by women’s ethnicity, education, age her periods began, family history of breast cancer, use of hormone replacement therapy, body-mass index, height, or consumption of alcohol or tobacco.

The authors conclude: “Use of oral contraceptives confers long-term protection against ovarian cancer. These findings suggest that oral contraceptives have already prevented some 200000 ovarian cancers and 100000 deaths from the disease, and that over the next few decades the numbers of cancers prevented will rise to at least 30000 per year.”

In an accompanying Comment, Dr Eduardo Franco, McGill University, Montreal, Quebec, Canada, and Dr Eliane Duarte-Franco, Institut National de Santé Publique de Québec, Montreal, Quebec, Canada, say: “As for the link between oral contraceptives and ovarian cancer, today’s collaborative analysis brings unequivocal good news. Women and their health-care providers are once again at a balancing act of judging risks versus benefits.”

And a linked Editorial in this week’s edition gives a risk-benefit overview of oral contraceptives and asks whether they should be made more widely available to women to protect them from ovarian cancer. It says: “We believe that the case is now convincing. Women deserve the choice to obtain oral contraceptives over-the-counter, removing a huge and unnecessary barrier to a potentially powerful cancer-preventing agent.”

It adds: “A strong message about the overall cancer preventing benefits of oral contraceptives would be a positive public-health message, empowering women to decide for themselves about the evidence.”

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