James M. Haughian

Cytokeratin 5 positive cells represent a steroid receptor negative and therapy resistant subpopulation in luminal breast cancers.

A majority of breast cancers are estrogen receptor (ER) positive and have a luminal epithelial phenotype. However, these ER+ tumors often contain heterogeneous subpopulations of ER− tumor cells. We previously identified a population of cytokeratin 5 (CK5) positive cells within ER+ and progesterone receptor positive (PR+) tumors that is both ER−PR− and CD44+, a marker of breast tumor-initiating cells (TICs). These CK5+ cells have properties of TICs in luminal tumor xenografts, and we speculated that they are more resistant to chemo- and anti-ER-targeted therapies than their ER+ neighbors. To test this, we used ER+PR+ T47D and MCF7 breast cancer cells. CK5+ cells had lower proliferative indices than CK5− cells, were less sensitive to 5-fluorouracil and docetaxel, and cultures became enriched for CK5+ cells after treatments. CK5+ cells were less prone to drug-induced apoptosis than CK5− cells. In cells treated with 17β-estradiol (E) plus anti-estrogens tamoxifen or fulvestrant, ER protein levels decreased, and CK5 protein levels increased, compared to controls treated with E alone. In ER+ tumors from patients treated with neoadjuvant endocrine therapies ER gene expression decreased, and CK5 gene expression increased in post compared to pre-treatment tumors. The number of CK5+ cells in tumors also increased in post- compared to pre-treatment tumors. We conclude that an ER−PR−CK5+ subpopulation found in many luminal tumors is resistant to standard endocrine and chemotherapies, relative to the majority ER+PR+CK5− cells. Compounds that effectively target these cells are needed to improve outcome in luminal breast cancers.

The Role of Luteinizing Hormone in Regulating Gene Expression During Selection of a Dominant Follicle in Cattle

At approximately 8.5 mm in diameter, the future dominant follicle is “selected” for continued growth in cattle. In the present study, cows were treated with a gonadotropin-releasing hormone receptor antagonist, acyline, just before follicle selection (near 7.8 mm) to investigate the role of LH in changing mRNA concentrations during selection of a dominant follicle. The ovaries containing the expected dominant follicle (EDF; first largest follicle) and expected largest subordinate follicle (ESF) were removed after 12 or 24 h of treatment. Real-time PCR was used to determine mRNA concentrations. ELISA was used to measure testosterone and 17beta-estradiol (E2) and radioimmunoassay to measure androstenedione (A4) in follicular fluid. Concentrations of E2 were greater in EDF than in ESF of untreated cows near the time of follicle selection (12 h) or at 12 h after selection (24 h). Testosterone, E2, and A4 were all dramatically decreased by acyline treatment at both times. In theca cells, acyline treatment reduced CYP17A1 (P450 17alpha) in EDF and STAR (steroidogenic acute regulatory protein) in both EDF and ESF but did not alter CYP11A1 (P450scc). In granulosa cells (GCs), LHCGR (luteinizing hormone [LH] receptor) was much greater in EDF than in ESF at both time of selection (739% greater) and 12 h after selection (2837% greater) and was decreased by acyline in EDF (87% decrease). The mRNA for CYP19A1 (cytochrome P450 aromatase) and PAPPA (pregnancy-associated plasma protein-A) tended to be greater in EDF than in ESF at follicle selection, and both mRNAs were much greater at 12 h after selection, with acyline significantly decreasing PAPPA mRNA after 24 h of treatment. The mRNA for FSHR (follicle-stimulating hormone receptor) was not different in EDF versus ESF and was not altered by acyline. Thus, induction of LHCGR mRNA in GCs is an early event during the follicle selection process, and surprisingly, expression of LHCGR mRNA is dependent on circulating LH. Production of follicular A4, testosterone, and E2 are also acutely related to LH but due to changes in expression of STAR and CYP17A1 in TC.

Maintenance of hormone responsiveness in luminal breast cancers by suppression of Notch

Luminal breast cancers express estrogen (ER) and/or progesterone (PR) receptors and respond to hormone therapies. Basal-like “triple negative” cancers lack steroid receptors but are cytokeratin (CK) 5-positive and require chemotherapy. Here we show that more than half of primary ER+PR+ breast cancers contain an ER−PR−CK5+ “luminobasal” subpopulation exceeding 1% of cells. Starting from ER+PR+ luminal cell lines, we generated lines with varying luminal to luminobasal cell ratios and studied their molecular and biological properties. In luminal disease, luminobasal cells expand in response to antiestrogen or estrogen withdrawal therapies. The phenotype and gene signature of the hormone-resistant cells matches that of clinical triple negative basal-like and claudin-low disease. Luminobasal cell expansion in response to hormone therapies is regulated by Notch1 signaling and can be blocked by γ-secretase inhibitors. Our data establish a previously unrecognized plasticity of ER+PR+ luminal breast cancers that, without genetic manipulation, mobilizes outgrowth of hormone-resistant basal-like disease in response to treatment. This undesirable outcome can be prevented by combining endocrine therapies with Notch inhibition.

Protein kinase C alpha-dependent signaling mediates endometrial cancer cell growth and tumorigenesis

Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage‐independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin‐dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homolog (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase‐3β (GSK‐3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting that PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of Grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK‐dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors.

Gonadotropin-releasing hormone, estradiol, and inhibin regulation of follicle-stimulating hormone and luteinizing hormone surges: implications for follicle emergence and selection in heifers.

ABSTRACT Mechanisms regulating gonadotropin surges and gonadotropin requirements for follicle emergence and selection were studied in heifers. Experiment 1 evaluated whether follicular inhibins regulate the preovulatory luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surges elicited by gonadotropin-releasing hormone (GnRH) injection (Hour = 0) and the subsequent periovulatory FSH surge. Treatments included control (n = 6), steroid-depleted bovine follicular fluid (bFF) at Hour −4 (n = 6), and bFF at Hour 6 (n = 6). Gonadotropins in blood were assessed hourly from Hours −6 to 36, and follicle growth tracked by ultrasound. Consistent with inhibin independence, bFF at Hour −4 did not impact the GnRH-induced preovulatory FSH surge, whereas treatment at Hour 6 delayed onset of the periovulatory FSH surge and impeded growth of a new follicular wave. Experiment 2 examined GnRH and estradiol (E2) regulation of the periovulatory FSH surge. Treatment groups were control (n = 8), GnRH-receptor antagonist (GnRHr-ant, n = 8), and E2 + GnRHr-ant (n = 4). GnRHr-ant (acyline) did not reduce the concentrations of FSH during the periovulatory surge and early follicle development (<7.0 mm) was unaffected, although subsequent growth of a dominant follicle (>8.0 mm) was prevented by GnRHr-ant. Addition of E2 delayed both the onset of the periovulatory FSH surge and emergence of a follicular wave. Failure to select a dominant follicle in the GnRHr-ant group was associated with reduced concentrations of LH but not FSH. Maximum diameter of F1 in controls (13.3 ± 0.5 mm) was greater than in both GnRHr-ant (7.7 ± 0.3 mm) and E2 + GnRHr-ant (6.7 ± 0.8 mm) groups. Results indicated that the periovulatory FSH surge stems from removal of negative stimuli (follicular E2 and inhibin), but is independent of GnRH stimulation. Emergence and early growth of follicles (until about 8 mm) requires the periovulatory FSH surge but not LH pulses. However, follicular deviation and late-stage growth of a single dominant follicle requires GnRH-dependent LH pulses.

Protein kinase C alpha (PKCα) regulates growth and invasion of endometrial cancer cells

The etiology of endometrial cancers remains poorly understood, particularly with respect to signal transduction pathways underlying the development and progression of the more aggressive, type II steroid‐independent tumors. Protein kinase C alpha (PKCα) regulates cellular processes critical to malignancy and has been implicated in the pathogenesis of endometrial cancers. The objective of these studies was to determine the functional role of PKCα in endometrial cancer cell proliferation, anchorage‐independent growth, and invasion. PKCα expression in endometrial cancer cell lines was examined by Western blotting. PKCα levels were increased in type II HEC‐50, HEC‐1‐A and HEC‐1‐B cell lines relative to the type I Ishikawa and RL‐95‐2 lines. Retroviral constructs were used to either overexpress PKCα or selectively knockdown levels by shRNA in Ishikawa and HEC 50 cells, respectively. Knockdown of PKCα expression in HEC‐50 cells resulted in a diminished growth rate and attenuation of anchorage‐independent growth. Correspondingly, Ishikawa cells overexpressing PKCα protein exhibited increased proliferation, resistance to growth factor deprivation and enhanced anchorage‐independent growth. Consistent with the observed changes in cell proliferation, PKCα also modulated cyclin D1 promoter activity in both cell lines. A reduction in PKCα levels rendered HEC‐50 cells significantly less invasive, whereas PKCα overexpression enhanced invasion of Ishikawa cells. These data indicate that PKCα promotes growth and invasion of endometrial cancer cells, suggesting that PKCα dependent signaling pathways could provide novel prognostic indicators or therapeutic targets, particularly in clinically aggressive type II endometrial tumors. J. Cell. Physiol. 220: 112–118, 2009.

Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells

IntroductionLuminal, estrogen receptor-positive (ER+) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models.MethodsImmunocompromised, ovariectomized (ovx’d) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER+PR+ cytokeratin 5-negative (CK5−) E3 and MCF-7 cells, basal ER−PR−CK5+ estrogen withdrawn-line 8 (EWD8) cells, and basal ER−PR−CK5− MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx’d mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers.ResultsER−PR− cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx’d mice injected with ER+PR+ cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER+PR+CK5− cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER−PR−CK5+ cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx’d control mice injected with ER+PR+ cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER+ but PR−. Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration.ConclusionsWe conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER−PR− subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx’d mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes.

c-Jun N-terminal kinase regulates apoptosis in endometrial cancer cells

Abstractc-Jun N-terminal kinases (JNKs) are important regulators of cell proliferation and apoptosis that have been implicated in tumorigenesis. We investigated the role of JNKs in apoptotic responses in Ishikawa and HEC-50 cells, models of type I and type II endometrial cancer, respectively. Etoposide treatment or UV irradiation resulted in sustained activation of JNK, correlating with the induction of apoptosis. Inhibition of JNK, or MAP kinase kinase 4 (MKK4), selectively suppressed apoptotic responses in both Ishikawa and HEC-50 cells. Knockdown of protein kinase C δ (PKCδ) also attenuated apoptosis in endometrial cancer cells and inhibited the sustained, UV-mediated JNK activation in HEC-50, but not Ishikawa cells. Etoposide-induced JNK phosphorylation was unaffected by PKCδ knockdown, implying that JNK can regulate apoptosis by PKCδ-dependent and independent pathways, according to stimulus and cell type. Thus, expression and activity of JNK and PKCδ in endometrial cancer cells modulate apoptosis and sensitivity to chemotherapeutic agents and may function as tumor suppressors in the endometrium.

Modeling luminal breast cancer heterogeneity: combination therapy to suppress a hormone receptor-negative, cytokeratin 5-positive subpopulation in luminal disease

IntroductionMany Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+ PR+ ones. One such subpopulation we call “Luminobasal” is ER-, PR- and cytokeratin 5 (CK5)-positive. It is not targeted for treatment.MethodsTo address the relationships between ER+PR+CK5– and ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We used high-throughput screening to identify pLB-specific drugs and examined their efficacy alone and in combination with hormone therapy in mixed-cell tumor models.ResultsWe show that pLUM and MCF7 cells suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and that pLUM cells suppress growth of pLB cells in mixed-cell xenografts in vivo. High-throughput screening of 89 FDA-approved oncology drugs shows that pLB cells are sensitive to monotherapy with the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib. By exploiting mixed-cell 3D colonies and mixed-cell solid mouse tumors models we demonstrate that combination therapy with gefitinib plus the anti-estrogen fulvestrant constitutes a robust treatment strategy.ConclusionsWe propose that response to combination endocrine/EGFR inhibitor therapies in heterogeneous Luminal cancers may improve long-term survival in patients whose primary tumors have been preselected for appropriate biomarkers, including ER, PR, CK5 and EGFR.

Abstract 1388: Luminal breast cancer metastasis is hormone-dependent and progesterone activates dormant disease.

Luminal, estrogen (ER) and/or progesterone (PR) receptor-positive breast cancers are the least aggressive forms of the disease. However, luminal cancers are the most common (70-80%) breast cancer subtype and therefore overall responsible for the greatest number of deaths due to metastases. The role of estrogens (E) in promoting breast cancer growth is well known. In contrast, little is known about the role of E or progesterone (P) in breast cancer metastasis. Most metastasis models use ER-PR- basal-like cells in which the role of women9s hormones cannot be studied. We have generated a metastasis model in ovariectomized immune- compromised mice using 100,000 human luminal or basal-like breast cancer cells injected directly into the arterial circulation. Tumor cells were simultaneously tagged with luciferase and ZsGreen to quantify whole-body metastatic burden in vivo and organ-specific metastases at necropsy. Mice were untreated (control), or supplemented with physiological concentrations of E or E+P (n=10/group). To avoid effects due to genetic variation, we used two new isogenic sublines of luminal T47D cells in our studies: E3, which are ER+PR+ and EWD8, which are identical but ER-PR-. We also tested luminal MCF-7 cells and basal MDA-231 cells. 1. Due to extensive metastases, survival of ER-PR- EWD8 or MDA-231-injected mice was brief (25-45 days) regardless of hormone treatment. 2. In contrast, luminal E3 or MCF-7-injected control mice appeared to be metastasis-free and were long-lived. However, E or E+P supplementation promoted luminal tumor metastasis. P was not protective; rather it tended to enhance metastatic burden, especially in E3 cells. 3. Major sites of luminal metastases in hormone-replete mice were bone, liver, lung and brain, analogous to sites seen clinically. 4. We performed a study in which mice injected with luminal tumor cells were maintained under control conditions for 8 weeks with no evidence of metastasis. They were then switched to E or E+P. E alone had no effect, but E+P activated metastasis in approximately 30% of mice. This suggests that a reservoir of dormant luminal metastatic cells remain sequestered in control tissues that under estrogenized conditions can be activated by P. 5. In luminal bone metastases, P significantly enhanced expression of cells that lack ER and PR, but express the basal-like maker cytokeratin 5 (CK5). The proliferation rate of the CK5+ subpopulation was markedly reduced compared to their ER+PR+ neighbors. Conclusions: E and P promote luminal breast cancer metastasis, may reactivate dormant disease at metastatic sites, and stimulate expression of an ER-PR- metastatic tumor-cell subpopulation that would be resistant to hormone therapies. Support: Department of Defense Breast Cancer Research Program, National Institute of Health CA026869-33, and the Breast Cancer Research Foundation. Citation Format: Ndiya Ogba, Nicole Manning, Brian Bliesner, Kelly Ambler, James Haughian, Mauricio Pinto, Kathryn B. Horwitz. Luminal breast cancer metastasis is hormone-dependent and progesterone activates dormant disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2013-1388