Kimberly R. Kalli

Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24(-/lo)CD44(+) phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.

Folate receptor alpha as a tumor target in epithelial ovarian cancer.

OBJECTIVES Folate receptor alpha (FRalpha) is a folate-binding protein overexpressed on ovarian and several other epithelial malignancies that can be used as a target for imaging and therapeutic strategies. The goal of this study is to improve historical data that lack specific information about FRalpha expression in rare histological subtypes, primary disease versus metastatic foci, and recurrent disease. METHODS FRalpha expression was analyzed by immunohistochemistry on 186 primary and 27 recurrent ovarian tumors, including 24 pairs of samples obtained from the same individuals at diagnosis and at secondary debulking surgery. For 20 of the 186 primaries, simultaneous metastatic foci were also analyzed. FRalpha staining was analyzed in light of disease morphology, stage, grade, debulking status, and time from diagnosis to recurrence and death. RESULTS FRalpha expression was apparent in 134 of 186 (72%) primary and 22 of 27 (81.5%) recurrent ovarian tumors. In 21 of 24 (87.5%) matched specimens, recurrent tumors reflected the FRalpha status detected at diagnosis. Metastatic foci were similar to primary tumors in FRalpha staining. FRalpha status was not associated with time to recurrence or overall survival in either univariate or multivariable analyses. CONCLUSION FRalpha expression occurs frequently, especially in the common high-grade, high-stage serous tumors that are most likely to recur. New findings from this study show that FRalpha expression is maintained on metastatic foci and recurrent tumors, suggesting that novel folate-targeted therapies may hold promise for the majority of women with either newly diagnosed or recurrent ovarian cancer.

In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417.

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.

Interleukin-1 signal transduction

Interleukin-1 (IL-1) is primarily an inflammatory cytokine, although it is capable of mediating a wide variety of effects on many different cell types. Nearly every known signal transduction pathway has been reported to be activated in response to IL-1. However, the significance of many of these signaling events is unclear, due to the use of different and sometimes unique cell lines in studying IL-1-initiated signal transduction. Complicating matters further is the lack of association in many studies between identified IL-1-induced signals and subsequent biological responses. In this article, we review what is known about IL-1 receptor signaling and, whenever possible, correlate signaling events to biological responses.

Hormone-receptor expression and ovarian cancer survival: an Ovarian Tumor Tissue Analysis consortium study

BACKGROUND Few biomarkers of ovarian cancer prognosis have been established, partly because subtype-specific associations might be obscured in studies combining all histopathological subtypes. We examined whether tumour expression of the progesterone receptor (PR) and oestrogen receptor (ER) was associated with subtype-specific survival. METHODS 12 studies participating in the Ovarian Tumor Tissue Analysis consortium contributed tissue microarray sections and clinical data to our study. Participants included in our analysis had been diagnosed with invasive serous, mucinous, endometrioid, or clear-cell carcinomas of the ovary. For a patient to be eligible, tissue microarrays, clinical follow-up data, age at diagnosis, and tumour grade and stage had to be available. Clinical data were obtained from medical records, cancer registries, death certificates, pathology reports, and review of histological slides. PR and ER statuses were assessed by central immunohistochemistry analysis done by masked pathologists. PR and ER staining was defined as negative (<1% tumour cell nuclei), weak (1 to <50%), or strong (≥50%). Associations with disease-specific survival were assessed. FINDINGS 2933 women with invasive epithelial ovarian cancer were included: 1742 with high-grade serous carcinoma, 110 with low-grade serous carcinoma, 207 with mucinous carcinoma, 484 with endometrioid carcinoma, and 390 with clear-cell carcinoma. PR expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p<0·0001) and high-grade serous carcinoma (log-rank p=0·0006), and ER expression was associated with improved disease-specific survival in endometrioid carcinoma (log-rank p<0·0001). We recorded no significant associations for mucinous, clear-cell, or low-grade serous carcinoma. Positive hormone-receptor expression (weak or strong staining for PR or ER, or both) was associated with significantly improved disease-specific survival in endometrioid carcinoma compared with negative hormone-receptor expression, independent of study site, age, stage, and grade (hazard ratio 0·33, 95% CI 0·21-0·51; p<0·0001). Strong PR expression was independently associated with improved disease-specific survival in high-grade serous carcinoma (0·71, 0·55-0·91; p=0·0080), but weak PR expression was not (1·02, 0·89-1·18; p=0·74). INTERPRETATION PR and ER are prognostic biomarkers for endometrioid and high-grade serous ovarian cancers. Clinical trials, stratified by subtype and biomarker status, are needed to establish whether hormone-receptor status predicts response to endocrine treatment, and whether it could guide personalised treatment for ovarian cancer. FUNDING Carraresi Foundation and others.

Expression of p16 and Retinoblastoma Determines Response to CDK4/6 Inhibition in Ovarian Cancer

Purpose: PD-0332991 is a selective inhibitor of the CDK4/6 kinases with the ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. Here we investigate the role of CDK4/6 inhibition in human ovarian cancer. Experimental Design: We examined the effects of PD-0332991 on proliferation, cell-cycle, apoptosis, and Rb phosphorylation using a panel of 40 established human ovarian cancer cell lines. Molecular markers for response prediction, including p16 and Rb, were studied using gene expression profiling, Western blot, and array CGH. Multiple drug effect analysis was used to study interactions with chemotherapeutic drugs. Expression of p16 and Rb was studied using immunohistochemistry in a large clinical cohort of ovarian cancer patients. Results: Concentration-dependent antiproliferative effects of PD-0332991 were seen in all ovarian cancer cell lines, but varied significantly between individual lines. Rb-proficient cell lines with low p16 expression were most responsive to CDK4/6 inhibition. Copy number variations of CDKN2A, RB, CCNE1, and CCND1 were associated with response to PD-0332991. CDK4/6 inhibition induced G0/G1 cell cycle arrest, blocked Rb phosphorylation in a concentration-and time-dependent manner, and enhanced the effects of chemotherapy. Rb-proficiency with low p16 expression was seen in 97/262 (37%) of ovarian cancer patients and was independently associated with poor progression-free survival (adjusted relative risk 1.49, 95% CI 1.00–2.24, P = 0.052). Conclusions: PD-0332991 shows promising biologic activity in ovarian cancer cell lines. Assessment of Rb and p16 expression may help select patients most likely to benefit from CDK4/6 inhibition in ovarian cancer. Clin Cancer Res; 17(6); 1591–602. ©2011 AACR.

Gene Expression Profiles Predict Early Relapse in Ovarian Cancer after Platinum-Paclitaxel Chemotherapy

Purpose: Women with advanced epithelial ovarian cancer are routinely treated with platinum-paclitaxel chemotherapy following cytoreductive surgery, yet only ∼20% achieve long-term disease-free survival. We hypothesized that differences in gene expression before treatment could distinguish patients with short versus long time to recurrence after administration of platinum-paclitaxel combination chemotherapy. Experimental Design: To test this hypothesis, gene expression profiling of 79 primary surgically resected tumors from women with advanced-stage, high-grade epithelial ovarian cancer was done using cDNA microarrays containing 30,721 genes. Supervised learning algorithms were applied in an effort to develop a binary classifier that could discriminate women at risk for early (≤21 months) versus late (>21 months) relapse after initial chemotherapy. Results: A 14-gene predictive model was developed using a set of training samples (n = 51) and subsequently tested using an independent set of test samples (n = 28). This model correctly predicted the outcome of 24 of the 28 test samples (86% accuracy) with 95% positive predictive value for early relapse. Conclusions: Predictive markers for early recurrence can be identified for platinum-paclitaxel combination chemotherapy in primary ovarian carcinoma. The proposed 14-gene model requires further validation.

Serine protease HtrA1 modulates chemotherapy-induced cytotoxicity

Resistance to chemotherapy presents a serious challenge in the successful treatment of various cancers and is mainly responsible for mortality associated with disseminated cancers. Here we show that expression of HtrA1, which is frequently downregulated in ovarian cancer, influences tumor response to chemotherapy by modulating chemotherapy-induced cytotoxicity. Downregulation of HtrA1 attenuated cisplatin- and paclitaxel-induced cytotoxicity, while forced expression of HtrA1 enhanced cisplatin- and paclitaxel-induced cytotoxicity. HtrA1 expression was upregulated by both cisplatin and paclitaxel treatment. This upregulation resulted in limited autoproteolysis and activation of HtrA1. Active HtrA1 induces cell death in a serine protease-dependent manner. The potential role of HtrA1 as a predictive factor of clinical response to chemotherapy was assessed in both ovarian and gastric cancer patients receiving cisplatin-based regimens. Patients with ovarian or gastric tumors expressing higher levels of HtrA1 showed a higher response rate compared with those with lower levels of HtrA1 expression. These findings uncover what we believe to be a novel pathway by which serine protease HtrA1 mediates paclitaxel- and cisplatin-induced cytotoxicity and suggest that loss of HtrA1 in ovarian and gastric cancers may contribute to in vivo chemoresistance.

Tumor-Infiltrating Programmed Death Receptor-1+ Dendritic Cells Mediate Immune Suppression in Ovarian Cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1+ B7-H1+ DCs have a classical DC phenotype (i.e., CD11c+CD11b+CD8−), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1+B7-H1+ DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1+B7-H1+ DCs in mediating immune suppression in ovarian cancer.

Prognostic and Therapeutic Relevance of Molecular Subtypes in High-Grade Serous Ovarian Cancer

Molecular classification of high-grade serous ovarian cancer (HGSOC) using transcriptional profiling has proven to be complex and difficult to validate across studies. We determined gene expression profiles of 174 well-annotated HGSOCs and demonstrate prognostic significance of the prespecified TCGA Network gene signatures. Furthermore, we confirm the presence of four HGSOC transcriptional subtypes using a de novo classification. Survival differed statistically significantly between de novo subtypes (log rank, P = .006) and was the best for the immunoreactive-like subtype, but statistically significantly worse for the proliferative- or mesenchymal-like subtypes (adjusted hazard ratio = 1.89, 95% confidence interval = 1.18 to 3.02, P = .008, and adjusted hazard ratio = 2.45, 95% confidence interval = 1.43 to 4.18, P = .001, respectively). More prognostic information was provided by the de novo than the TCGA classification (Likelihood Ratio tests, P = .003 and P = .04, respectively). All statistical tests were two-sided. These findings were replicated in an external data set of 185 HGSOCs and confirm the presence of four prognostically relevant molecular subtypes that have the potential to guide therapy decisions.