Deanna M. Janzen

Stem‐Like Epithelial Cells Are Concentrated in the Distal End of the Fallopian Tube: A Site for Injury and Serous Cancer Initiation

The reproductive role of the fallopian tube is to transport the sperm and egg. The tube is positioned to act as a bridge between the ovary where the egg is released and the uterus where implantation occurs. Throughout reproductive years, the fallopian tube epithelium undergoes repetitive damage and regeneration. Although a reservoir of adult epithelial stem cells must exist to replenish damaged cells, they remain unidentified. Here, we report isolation of a subset of basally located human fallopian tube epithelia (FTE) that lack markers of ciliated (β‐tubulin; TUBB4) or secretory (PAX8) differentiated cells. These undifferentiated cells expressed cell surface antigens: epithelial cell adhesion molecule, CD44, and integrin α 6. This FTE subpopulation was fivefold enriched for cells capable of clonal growth and self‐renewal suggesting that they contain the FTE stem‐like cells (FTESCs). A twofold enrichment of the FTESC was found in the distal compared to the proximal end of the tube. The distal fimbriated end of the fallopian tube is a well‐characterized locus for initiation of serous carcinomas. An expansion of the cells expressing markers of FTESC was detected in tubal intraepithelial carcinomas and in fallopian tubes from patients with invasive serous cancer. These findings suggest that FTESC may play a role in the initiation of serous tumors. Characterization of these stem‐like cells will provide new insight into how the FTE regenerate, respond to injury, and may initiate cancer. STEM CELLS2012;30:2487–2497

Cell-autonomous activation of the PI3-kinase pathway initiates endometrial cancer from adult uterine epithelium

Epithelial-specific activation of the PI3-kinase pathway is the most common genetic alteration in type I endometrial cancer. In the majority of these tumors, PTEN expression is lost in the epithelium but maintained in tumor stroma. Currently reported PTEN knockout mouse models initiate type I endometrial cancer concomitant with loss of PTEN in both uterine epithelium and stroma. Consequently, the biologic outcome of selectively activating the PI3-kinase pathway in the endometrial epithelium remains unknown. To address this question, we established a malleable in vivo endometrial regeneration system from dissociated murine uterine epithelium and stroma. Regenerated endometrial glands responded to pharmacologic variations in hormonal milieu similar to the native endometrium. Cell-autonomous activation of the PI3-kinase pathway via biallelic loss of PTEN or activation of AKT in adult uterine epithelia in this model was sufficient to initiate endometrial carcinoma. AKT-initiated tumors were serially transplantable, demonstrating permanent genetic changes in uterine epithelia. Immunohistochemistry confirmed loss of PTEN or activation of AKT in regenerated hyperplastic glands that were surrounded by wild-type stroma. We demonstrate that cell-autonomous activation of the PI3-kinase pathway is sufficient for the initiation of endometrial carcinoma in naive adult uterine epithelia. This in vivo model provides an ideal platform for testing the response of endometrial carcinoma to targeted therapy against this common genetic alteration.

Role of autonomous androgen receptor signaling in prostate cancer initiation is dichotomous and depends on the oncogenic signal

The steroid hormone signaling axis is thought to play a central role in initiation and progression of many hormonally regulated epithelial tumors. It is unclear whether all cancer-initiating signals depend on an intact hormone receptor signaling machinery. To ascertain whether cell autonomous androgen receptor (AR) is essential for initiation of prostate intraepithelial neoplasia (PIN), the response of AR-null prostate epithelia to paracrine and cell autonomous oncogenic signals was assessed in vivo by using the prostate regeneration model system. Epithelial-specific loss of AR blocked paracrine FGF10-induced PIN, whereas the add back of exogenous AR restored this response. In contrast, PIN initiated by cell-autonomous, chronic-activated AKT developed independent of epithelial AR signaling. Our findings demonstrate a selective role for AR in the initiation of PIN, dependent on the signaling pathways driving tumor formation. Insights into the role of hormone receptor signaling in the initiation of epithelial tumors may help define this axis as a target for chemoprevention of carcinomas.

An apoptosis-enhancing drug overcomes platinum resistance in a tumour-initiating subpopulation of ovarian cancer.

High-grade serous ovarian cancers (HGSCs) are deadly malignancies that relapse despite carboplatin chemotherapy. Here we show that 16 independent primary HGSC samples contain a CA125-negative population enriched for carboplatin-resistant cancer initiating cells. Transcriptome analysis reveals upregulation of homologous recombination DNA repair and anti-apoptotic signals in this population. While treatment with carboplatin enriches for CA125-negative cells, co-treatment with carboplatin and birinapant eliminates these cells in HGSCs expressing high levels of the inhibitor of apoptosis protein cIAP in the CA125-negative population. Birinapant sensitizes CA125-negative cells to carboplatin by mediating degradation of cIAP causing cleavage of caspase 8 and restoration of apoptosis. This co-therapy significantly improves disease-free survival in vivo compared with either therapy alone in tumour-bearing mice. These findings suggest that therapeutic strategies that target CA125-negative cells may be useful in the treatment of HGSC. Despite normalization of the CA125 serum biomarker at the completion of carboplatin therapy the vast majority of patients with high grade serous ovarian cancers relapse. Here, Janzen et al., identify a sub-population of tumor cells that are CA125 negative, cancer initiating and platinum resistant but readily eliminated with the addition of apoptosis enhancing drugs to carboplatin.

Progesterone Receptor Signaling in the Microenvironment of Endometrial Cancer Influences Its Response to Hormonal Therapy

Progesterone, an agonist for the progesterone receptor (PR), can be an efficacious and well-tolerated treatment in endometrial cancer. The clinical use of progesterone is limited because of the lack of biomarkers that predict hormone sensitivity. Despite its efficacy in cancer therapy, mechanisms and site of action for progesterone remain unknown. Using an in vivo endometrial cancer mouse model driven by clinically relevant genetic changes but dichotomous responses to hormonal therapy, we show that signaling through stromal PR is necessary and sufficient for progesterone antitumor effects. Endometrial cancers resulting from epithelial loss of PTEN (PTENKO) were hormone sensitive and had abundant expression of stromal PR. Stromal deletion of PR as a single genetic change in these tumors induced progesterone resistance indicating that paracrine signaling through the stroma is essential for the progesterone therapeutic effects. A hormone-refractory endometrial tumor with low levels of stromal PR developed when activation of KRAS was coupled with PTEN-loss (PTENKO/Kras). The innate progesterone resistance in PTENKO/Kras tumors stemmed from methylation of PR in the tumor microenvironment. Add-back of stromal PR expressed from a constitutively active promoter sensitized these tumors to progesterone therapy. Results show that signaling through stromal PR is sufficient for inducing hormone responsiveness. Our findings suggest that epigenetic derepression of stromal PR could be a potential therapeutic target for sensitizing hormone-refractory endometrial tumors to progesterone therapy. On the basis of these results, stromal expression of PR may emerge as a reliable biomarker in predicting response to hormonal therapy.

Estrogen and progesterone together expand murine endometrial epithelial progenitor cells.

Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here, we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multilineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Coadministration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore, their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium. STEM CELLS 2013;31:808–822

Low Levels of Circulating Estrogen Sensitize PTEN-null Endometrial Tumors to PARP Inhibition In Vivo

Earlier in vitro work demonstrated that PARP inhibition induces cell death in PTEN-null endometrial cancer cell lines, but the in vivo therapeutic efficacy of these agents against endometrial cancer remains unknown. Here, we test the efficacy of AZD2281 (olaparib), an oral PARP inhibitor, in the therapy of PTEN-null endometrial tumors in a preclinical endometrial cancer mouse model. Primary endometrial tumors were generated by epithelial loss of PTEN using an in vivo model. This model recapitulates epithelial-specific loss of PTEN seen in human tumors, and histologically resembles endometrioid carcinomas, the predominant subtype of human endometrial cancers. Olaparib was administered orally to tumor-bearing mice in two hormonal extremes: high or low estrogen. Olaparib treatment achieved a significant reduction in tumor size in a low estrogenic milieu. In striking contrast, no response to olaparib was seen in tumors exposed to high levels of estrogen. Two key observations were made when estrogen levels were dropped: (i) the serum concentration of olaparib was significantly increased, resulting in sustained PARP inhibition at the tumor bed; and (ii) the homologous recombination pathway was compromised, as evidenced by decreased Rad51 protein expression and function. These two mechanisms may account for the sensitization of PTEN-null tumors to olaparib with estrogen deprivation. Results of this preclinical trial suggest that orally administered PARP inhibitors in a low estrogenic hormonal milieu can effectively target PTEN-null endometrial tumors. Extension of this work to clinical trials could personalize the therapy of women afflicted with advanced endometrial cancer using well-tolerated orally administered therapeutic agents. Mol Cancer Ther; 12(12); 2917–28. ©2013 AACR.

Birinapant sensitizes platinum-resistant carcinomas with high levels of cIAP to carboplatin therapy

Platinum drugs are the frontline therapy in many carcinomas, including high-grade serous ovarian cancers. Clinically, high-grade serous carcinomas have an apparent complete response to carboplatin, but tumors invariably recur and response to platinum drugs diminishes over time. Standard of care prohibits re-administration of platinum drugs to these patients who are labeled as having platinum-resistant disease. In this stage patients are treated with non-platinum agents and outcomes are often poor. In vivo and in vitro data presented here demonstrate that this clinical dogma should be challenged. Platinum drugs can be an effective therapy even for platinum-resistant carcinomas as long as they are combined with an agent that specifically targets mechanisms of platinum resistance exploited by the therapy-resistant tumor subpopulations. High levels of cellular inhibitor of apoptosis proteins cIAP1 and 2 (cIAP) were detected in up to 50% of high-grade serous and non-high-grade serous platinum-resistant carcinomas. cIAP proteins can induce platinum resistance and they are effectively degraded with the drug birinapant. In platinum-resistant tumors with ≥22.4 ng of cIAP per 20 µg of tumor lysate, the combination of birinapant with carboplatin was effective in eliminating the cancer. Our findings provide a new personalized therapeutic option for patients with platinum-resistant carcinomas. The efficacy of birinapant in combination with carboplatin should be tested in high-grade serous carcinoma patients in a clinical trial.Combination therapy: sensitizing platinum-resistant tumorsCarboplatin efficacy can be restored by administering it with birinapant, a drug combination that targets platinum-resistant cancer cells. Platinum-based chemotherapeutics are used to treat almost 50% of cancer patients. Despite excellent initial response rates, tumor growth recurs in most patients as they develop resistance to therapy. Sanaz Memarzadeh and colleagues at the University of California, Los Angeles, show that the addition of birinapant to carboplatin eliminates platinum-resistant ovarian cancer cells by degrading the cell death-inhibiting proteins cIAP1 and cIAP2. Carboplatin and birinapant co-therapy restored the efficacy of the chemotherapeutic in human tumor samples and in mice with platinum-resistant tumors. Furthermore, the levels of cIAP protein in the resistant cells were predictive of the therapeutic response. These findings will help to select clinical trial patients that are most likely to benefit from this combination therapy.

Tubal Ligation Induces Quiescence in the Epithelia of the Fallopian Tube Fimbria

Tubal ligation keeps the fimbriated end of the fallopian tube intact while interrupting the conduit for sperm and egg between the uterus and ovary. Tubal ligation is associated with an approximately 20% decreased risk of high-grade serous ovarian cancers, which mounting evidence suggests arise from the distal fallopian tube epithelium. We postulated that biological changes at the epithelial cellular level of the distal fallopian tube may account for the surgical procedure’s observed risk reduction. We compared the histology, presence of epithelial progenitors (basally located CD44-positive cells), and degree of epithelial proliferation (Ki67-positive cells) of distal fallopian tube from 10 patients with previous tubal ligation and 10 age-matched patients with uncut fallopian tubes. A significantly reduced population of proliferating epithelial progenitors (basally located CD44/Ki67 dual-positive cells) was detected in the tubal ligated specimens (P = .0002). To functionally assess the effect of tubal ligation, a murine model was utilized to compare the growth capacity of distal fallopian tube epithelial cells isolated from either ligated or sham-operated tubal epithelia. Murine fallopian tube epithelial cells isolated after tubal ligation showed a significantly reduced capacity to grow organoids in culture compared to sham-operated controls (P = .002). The findings of this study show that tubal ligation is associated with a reduced presence and decreased proliferation of progenitor cells in the distal fallopian tube epithelium. These compositional and functional changes suggest that tubal ligation induces quiescence of distal fallopian tube epithelial cells.

Miniring approach for high-throughput drug screenings in 3D tumor models

There is increasing interest in developing 3D tumor organoid models for drug development and personalized medicine applications. While tumor organoids are in principle amenable to high-throughput drug screenings, progress has been hampered by technical constraints and extensive manipulations required by current methodologies. Here, we introduce a miniaturized, fully automatable, flexible high-throughput method using a simplified geometry to rapidly establish 3D organoids from cell lines and primary tissue and robustly assay drug responses.There is increasing interest in developing 3D tumor organoid models for drug development and personalized medicine approaches. While tumor organoids are in principle amenable to high-throughput drug screenings, progress has been hampered by technical constraints and extensive manipulations required by current approaches. Here, we introduce a miniaturized, fully automatable, flexible high-throughput method using a simplified geometry to establish 3D organoids from cell lines and primary tumors and robustly assay drug responses.