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Background PD-1 inhibitors have shown significant efficacy in triple negative breast cancer (BC), however, durable responses are less common in estrogen receptor-positive (ER+) BC. Better markers are therefore needed that will identify likely responders to PD-1 inhibitors among patients with luminal BC. While most efforts have focused on the immune checkpoint protein PD-L1, the alternative PD-1 ligand, PD-L2, has been largely overlooked. We aimed to determine if PD-L2 is associated with unfavorable prognosis in ER+ BC. Methods PD-L2 protein levels in cancer cells and stromal cells were measured retrospectively by quantitative immunofluorescence histocytometry in tissue microarrays of therapy-naïve, localized or locoregional ER+ BC and correlated with progression-free survival (PFS). Evaluable tumor PD-L2 data were derived from a main study cohort A diagnosed between 1988-2005 (n=684) with extensive clinical and outcome data and from an independent validation cohort B diagnosed between 1992-2012 (n=273). Patients received standard-of-care adjuvant therapy without immune checkpoint inhibitors after tumor resection. Results Univariate analysis of the main cohort A revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (HR=1.8; 95%CI:1.3-2.6; p=0.001), an observation that was validated in an independent cohort B (HR=2.3, 95%CI:1.1-4.8; p=0.026). Approximately one third of ER+ BC cases were classified as high PD-L2. After multivariable adjustment for common clinicopathological variables, high cancer cell levels of PD-L2 remained independently predictive of early recurrence (HR=2.0; 95%CI:1.4-2.9; p< 0.001). Sub-analysis of ER+ BC cases treated with adjuvant chemotherapy (n=197) suggested that high PD-L2 levels in cancer cells was associated with particularly increased risk of progression (multivariable HR=3.4; 95%CI:1.9-6.2; p< 0.001). The observed frequent expression of PD-L2 protein in BC provided scientific rationale for the design of our ongoing phase II clinical trial (NCT04243616) of neoadjuvant combined PD-1 inhibitor (cemiplimab; Regeneron Pharmaceuticals Inc) and chemotherapy in patients diagnosed with PD-L1+ and/or PD-L2+ BC. The primary objective of this trial is to assess pathologic responses to neoadjuvant treatment with secondary objective of assessing the correlation between PD-L1/PD-L2 status and tumor responses. Pathologist review of PD-L1 and PD-L2 expression in an initial set of ER+ tumors (n=15) screened for trial eligibility revealed frequent discordance between cancer cell positivity for PD-L1 and PD-L2 protein (PD-L1: median=0%; range 0-2% vs. PD-L2: median=18%; range < 1-60%) as well as immune cell positivity (PD-L1: median=5%; range 0-50% vs. PD-L2: median=0; range 0-50%). Conclusions In treatment-naïve ER+ breast tumors, high cancer cell expression of PD-L2 protein was an independent predictor of poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Preliminary analyses of ER+ tumors from our ongoing clinical trial showed frequent discordance between baseline PD-L1 and PD-L2 protein expression in both cancer cells and immune cells. Collectively, our analyses indicate that PD-L2 has prognostic value for ER+ BC, and our progress justify further studies to determine whether PD-L2, alone or in combination with PD-L1, may serve as a predictive marker of response to PD-1 inhibitors. Citation Format: Lubna N. Chaudhary, Inna Chervoneva, Amy R. Peck, Yunguang Sun, Misung Yi, John F. Langenheim, Julie M. Jorns, Sailaja Kamaraju, Yee Chung Cheng, John Burfeind, Christopher R. Chitambar, Jeffrey A. Hooke, Albert J. Kovatich, Craig Shriver, Hai Hu, Juan P. Palazzo, Marluce Bibbo, Terry Hyslop, Richard Pestell, Edith P. Mitchell, Hallgeir Rui. High PD-L2 Protein Expression in Cancer Cells is an Independent Marker of Unfavorable Prognosis in Luminal Breast Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-13.

Background. Endocrine resistant metastatic disease develops in ~20-25% of hormone-receptor positive (HR+) breast cancer (BC) patients despite endocrine therapy (ET) use. Upregulation of HER family receptor tyrosine kinases (RTKs) represent escape mechanisms in response to ET in some HR+ tumors. Short-term neoadjuvant ET (NET) offers the opportunity to identify early endocrine escape mechanisms initiated in individual tumors. Methods. This was a single arm, interventional phase II clinical trial evaluating 4 weeks (+/-1 week) of NET in patients with early-stage HR+/HER2-negative (HER2-) BC. The primary objective was to assess NET-induced changes in HER1-4 proteins by immunohistochemistry (IHC) score. Protein upregulation was defined as an increase of ≥1 in IHC score following NET. Results. Thirty-seven patients with cT1-T3, cN0, HR+/HER2- BC were enrolled. In 35 patients with evaluable tumor HER protein after NET, HER2 was upregulated in 48.6% (17/35; p=0.025), with HER2-positive status (IHC 3+ or FISH-amplified) detected in three patients at surgery, who were recommended adjuvant trastuzumab-based therapy. Downregulation of HER3 and/or HER4 protein was detected in 54.2% of tumors, whereas HER1 protein remained low and unchanged in all cases. While no significant volumetric reduction was detected radiographically after short-term NET, significant reduction in tumor proliferation rates were observed. No significant associations were identified between any clinicopathologic covariates and changes in HER1-4 protein expression on multivariable analysis. Conclusion. Short-term NET frequently and preferentially upregulates HER2 over other HER-family RTKs in early-stage HR+/HER2- BC and may be a promising strategy to identify tumors that utilize HER2 as an early endocrine escape pathway. Trial registration number: NCT03219476 Date of registration for prospectively registered trials: July 17, 2017

Supplementary Table 1: List of oligonucleotides used for qPCR

S1. Frequency of Pan-IN lesion types in KC versus KCC mice. S2. Re-expression of Ch25h in PDAC cell lines. S3. Presence of exogenous lipids diminishes effect of Ch25h re-expression in PDAC. S4. Re-expression of Ch25h does not affect Ras localization. S5. CH25H decreases autophagic flux in PDAC cells. S6. Ch25h slows PDAC tumor growth.

Supplementary Table 4: Genes down-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Methods. Supplementary Figure 1. Chemical structures of the small molecule IST5-002 analogs. Supplementary Figure 2. IST5-002 inhibits phosphorylation of Stat5 in mouse and human breast cancer cells. Supplementary Figure 3. IST5-002 does not have significant inhibitory activity against 50 kinases tested. Supplementary Figure 4. IST5-002 does not affect phosphorylation of Pak1/2. Supplementary Figure 5. IST5-002 inhibits expression of Stat5 target genes in PC cells and in CML cells. Supplementary Figure 6. IST5-002 is not generally cytotoxic as determined by lack of cell death induction in cancer cell lines established from other organs. Supplementary Figure 7. CWR22Rv1 PC cells were inoculated subcutaneously into the flanks of castrated athymic nude mice supplied with sustained-release 5alpha-dihydrotestosterone (DHT)-pellets (n=10/group, 1 tumor/mouse, 1.5 ??107 CWR22Rv1 cells per site, 1 DHT pellet/mouse). Supplementary Figure 8. IST5-0-02 induces apoptosis in CWR22Rv1 tumors grown in nude mice and in clinical PCs cultured ex vivo in organ explant cultures. Supplementary Figure 9. IST5-002 induces extensive apoptotic death of imatinib-sensitive and -resistant CML cells.

Supplementary Table 3: Genes up-regulated (> 2-fold) by Stat5-shRNA and IST5-002 in K562

Supplementary Table 5: IST5-002 regulation of known Stat5 target genes in K562 cells.

Supplementary Table 2: Genes regulated by both Stat5-shRNA and IST5-002 in K562 cells, corresponding to the heatmap in Figure 2g.

Supplemental Table 1: Structure-Activity-Relationship analysis.

Cholesterol dependence is an essential characteristic of pancreatic ductal adenocarcinoma (PDAC). Cholesterol 25-hydroxylase (CH25H) catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol, which is implicated in inhibiting cholesterol biosynthesis and in cholesterol depletion. Here, we show that, within PDAC cells, accumulation of cholesterol was facilitated by the loss of CH25H. Methylation of the CH25H gene and decreased levels of CH25H expression occurred in human pancreatic cancers and was associated with poor prognosis. Knockout of Ch25h in mice accelerated progression of Kras-driven pancreatic intraepithelial neoplasia. Conversely, restoration of CH25H expression in human and mouse PDAC cells decreased their viability under conditions of cholesterol deficit, and decelerated tumor growth in immune competent hosts. Mechanistically, the loss of CH25H promoted autophagy resulting in downregulation of MHC-I and decreased CD8+ T-cell tumor infiltration. Re-expression of CH25H in PDAC cells combined with immune checkpoint inhibitors notably inhibited tumor growth. We discuss additional benefits that PDAC cells might gain from inactivation of CH25H and the potential translational importance of these findings for therapeutic approaches to PDAC.Implications:Loss of CH25H by pancreatic cancer cells may stimulate tumor progression and interfere with immunotherapies.

Background C-C Chemokine Ligand type-5 (CCR5) is overexpressed in >95% of TNBC and has been correlated with disease progression. Moreover, enhancement of DNA repair signaling by CCR5 activation may contribute to chemotherapy resistance. Therefore, blocking CCR5 may result in increased immune response against tumor cells and synergize with chemotherapy. Leronlimab (PRO 140) is a humanized monoclonal antibody to CCR5. Preclinical data showed leronlimab binds human CCR5, blocks CCR5-mediated signaling, and CCL5-induced breast cancer cell invasion. The therapeutic antibody leronlimab has been administered to over 800 healthy and HIV-1 infected individuals with good tolerance and without obvious dose-related toxicity, making it an ideal partner for chemotherapy combinations in TNBC. Methods In this ongoing phase 1B/2 study, patients with CCR5+ mTNBC with ≤2 line of therapy in the metastatic setting (no prior carboplatin) are treated with weekly subcutaneous leronlimab (3 dose levels, 3+3 dose escalation) and a fixed dose of carboplatin AUC 5 on day 1 with a 21-day dose-limiting toxicity (DLT) window, followed by expansion in 30 patients with CCR5+ mTNBC who are naïve to chemotherapy in the metastatic setting or who have failed first-line combination of chemotherapy (excluding carboplatin) and a checkpoint inhibitor in the metastatic setting. CCR5 positivity is centrally assessed by IHC and defined as >10% CCR5 staining in primary or metastatic tumor cells and/or high predominance of CCR5+ tumor-infiltrating leukocytes (TIL). Primary objectives are safety, tolerability, determination of maximum tolerated dose, and determination of the recommended phase 2 dose (RP2D). Results Fifteen patients had archived tumor tissue assessed for CCR5 expression, with 12 being CCR5 positive (median expression 20%, range 0 - 100%, and 7 out of 12 high CCR5+ TILs) . A total of ten patients (median age 51 years; median 2 prior therapies) have been enrolled at 3 dose levels (350, 525, and 700 mg). In the second cohort, 1 additional patient was inadvertently enrolled. All patients completed the DLT assessment period and no DLTs have been observed. Patients received between 3 and 27 doses of leronlimab with the number of cycles ranging from 1 to 9. Five patients remain on treatment. The most common treatment-emergent adverse events (TEAEs) by any grade were: fatigue (6/10), headache (4/10), constipation (3/10), and nausea (3/10). The following grade ≥3 TEAEs were reported: neutropenia, anemia, thrombocytopenia, hyponatremia, hypertension, diarrhea and headache. Serious Adverse Events were reported in 2 patients (grade 2 sepsis and grade 3 headache). The following leronlimab treatment related adverse events (TRAEs) occurred (all grade 1): injection site reaction in cohort 1, fatigue (n=2) and headache in cohort 2. Three carboplatin TRAEs ≥3 were reported in one patient in cohort 1: thrombocytopenia, anemia and leukopenia. Two out of seven patients eligible for response achieved a confirmed partial response, and 4 patients stable disease. Conclusions Leronlimab, in combination with carboplatin, has been well-tolerated in all 3 dose levels with early signs of anti-tumor activity in patients with CCR5+ mTNBC. The study is currently enrolling patients at the RP2D dose of leronlimab 700mg in combination with carboplatin AUC 5 in the phase 2 part of the trial. Clinical trial information: NCT03838367 Citation Format: Massimo Cristofanilli, Namita Chittoria, Sima Ehsani, Hallgeir Rui, Milana Dolezal, Lisette Stork-Sloots, Femke de Snoo, Christopher Recknor, Vandana Abramson. A phase Ib/II study of leronlimab combined with carboplatin in patients with CCR5+ metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-08.

Background Antiprogestins, including selective progesterone receptor (PgR) modulators (SPRMs) that act as PgR antagonists, are a promising class of therapeutics for overcoming endocrine resistance including in patients who develop activating estrogen receptor 1 (ESR1) mutations after prior endocrine therapy (ET). The SMILE study (NCT04738292) is a multi-institutional phase II clinical trial to determine the efficacy and safety of an antiprogestin, onapristone in combination with fulvestrant as second-line therapy for patients with ER+,PgR+, HER2 -metastatic breast cancer (MBC). Methods Patients with locally advanced or MBC who progress on ≥2 lines of prior therapy with ET as a single agent or in combination with CDK4/6 inhibitors, mTOR inhibitors and one line of chemotherapy in the metastatic setting are eligible. Other criteria include ECOG performance status ≤ 2, measurable disease per RECIST 1.1 criteria, adequate organ function, availability of an archived tumor biopsy block confirming the diagnosis of MBC, optional biopsy at disease progression on trial, and optional 18F-fluorofuranylnorprogesterone (18F-FFNP) PET/CT imaging. Ovarian suppression is allowed. Prior ET with tamoxifen or aromatase inhibitor therapy in the adjuvant or metastatic setting is allowed. Patients with prior exposure to fulvestrant, antiprogestins, or CDK inhibitors in the adjuvant setting are ineligible. Following consent, all subjects will receive fulvestrant at the recommended dosing schedule plus onapristone 50 mg orally, twice daily, until disease progression, unacceptable toxicity, or discontinuation for any other reason. Using the Simon Two-Stage design, a total of 39 patients will be enrolled over two years from 6-8 sites within Wisconsin Oncology Network (WON). In the first stage of the trial, 21 subjects will be enrolled; if ≥2 responses are observed, then 18 additional subjects will be enrolled in the second stage. This design yields a one-sided type I error rate of 5% if the ORR is 7% and power of 80% when the combination's true response rate is 20%. The primary objective is to evaluate the objective response rate; secondary objectives include safety and tolerability, progression-free survival, disease control rate, and duration of response. Other correlates include optional functional imaging of PgR binding with 18F-FFNP PET/CT and biomarker analysis (ER, total PgR, HER2, Ki67, CD24, CD44, LDH1, KLF4, CK 5/6, PhosphoSer294-PgR on tissue samples, ESR1 mutations, and circulating tumor DNA analysis). The study was approved in January 2021, and enrollment is active at the time of this submission. Citation Format: Sailaja Kamaraju, Amy Fowler, Chung Yee Cheng, Lubna N Chaudhary, John Burfeind, Janet Retseck, Amye J Tevaarwerk, Mark Burkard, Elisavet Paplomata, Amanda M Parkes, Julie M Jorns, Sergey Tarima, Douglas Yee, Hallgeir Rui, Carol Lange, Tarek Sahmoud, Kari B Wisinski. The SMILE study: A phase 2 trial of onapristone in combination with fulvestrant for patients with ER+ and HER2- metastatic breast cancer after progression on endocrine therapy and CDK4/6 inhibitors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-16-01.

Supplemental figure 1-11 which include: (1) High resolution image of figure 1A. (2) Supplemental data for the relation between CCR5+ cells and breast cancer stem cells. (3) In vivo tumor formation of CCR5+ vs CCR5- cells. (4) In vivo tumor formation of CCR5 overexpression cells vs vector control cells. (5) Gene expression profile of CCR5+ vs CCR5- cells. (6) The effects of CCR5 on DNA damage repair in MDA-MB-231 cells. (7) A schematic representation of the DNA damage signaling pathways, with target genes enriched in CCR5+ cells. (8) The effects of CCR5 on DNA damage repair based on DNA damage repair reporter assays. (9) The functional synergy of cell killing between CCR5 inhibitors and Doxorubicin. (10) Single cell RNAseq of CCR5+ vs. CCR5- SUM-159 cells. (11) Enrichment of CCR5+ cells in doxorubicin resistant cells.

Supplementary Data from Cyclin D1 Splice Variants: Polymorphism, Risk, and Isoform-Specific Regulation in Prostate Cancer

Supplemental table 1 and 2 is the clinical characteristics of patients analyzed in figure 1B. Supplemental table 3 is the GR50 of breast cancer cell lines treated with Doxorubicin with the addition of either Maraviroc or Vicriviroc.

Supplementary Figure 7: Schematic representation of structure of wildtype Stat5a and Stat5b, dominant-negative Stat5 (DN-Stat5) and constitutively-active Stat5 (CA-Stat5) proteins.

Supplementary Figure 1 Legend

Supplementary Figure 1 - Time to recurrence in stage I (blue), II (yellow), and III (black) patients with colorectal cancer.

The functional significance of the chemokine receptor CCR5 in human breast cancer epithelial cells is poorly understood. Here, we report that CCR5 expression in human breast cancer correlates with poor outcome. CCR5+ breast cancer epithelial cells formed mammospheres and initiated tumors with >60-fold greater efficiency in mice. Reintroduction of CCR5 expression into CCR5-negative breast cancer cells promoted tumor metastases and induced DNA repair gene expression and activity. CCR5 antagonists Maraviroc and Vicriviroc dramatically enhanced cell killing mediated by DNA-damaging chemotherapeutic agents. Single-cell analysis revealed CCR5 governs PI3K/Akt, ribosomal biogenesis, and cell survival signaling. As CCR5 augments DNA repair and is reexpressed selectively on cancerous, but not normal breast epithelial cells, CCR5 inhibitors may enhance the tumor-specific activities of DNA damage response–based treatments, allowing a dose reduction of standard chemotherapy and radiation.Significance: This study offers a preclinical rationale to reposition CCR5 inhibitors to improve the treatment of breast cancer, based on their ability to enhance the tumor-specific activities of DNA-damaging chemotherapies administered in that disease. Cancer Res; 78(7); 1657–71. ©2018 AACR.

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged.Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies.Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism.Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. (Clin Cancer Res 2009;15(17):5338–49)

Supplementary Figure S1.

Combined Supplementary Figures, Supplementary Tables, Legends and Key Resource Tables

The figure legends of Supplemental figure 1-11 which include: (1) High resolution image of figure 1A. (2) Supplemental data for the relation between CCR5+ cells and breast cancer stem cells. (3) In vivo tumor formation of CCR5+ vs CCR5- cells. (4) In vivo tumor formation of CCR5 overexpression cells vs vector control cells. (5) Gene expression profile of CCR5+ vs CCR5- cells. (6) The effects of CCR5 on DNA damage repair in MDA-MB-231 cells. (7) A schematic representation of the DNA damage signaling pathways, with target genes enriched in CCR5+ cells. (8) The effects of CCR5 on DNA damage repair based on DNA damage repair reporter assays. (9) The functional synergy of cell killing between CCR5 inhibitors and Doxorubicin. (10) Single cell RNAseq of CCR5+ vs. CCR5- SUM-159 cells. (11) Enrichment of CCR5+ cells in doxorubicin resistant cells.

Clinical Characteristics of Outcome Cohorts 1 and 2.

Supplementary Material-1. Heatmap shows all the known ligand-receptor interactions between different cell types identified by CellPhoneDB in the OvD5-SS2 dataset.

SupplementaryMethods.docx from Oncostatin M Receptor–Targeted Antibodies Suppress STAT3 Signaling and Inhibit Ovarian Cancer Growth

Purpose:Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing.Experimental Design:We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome.Results:PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome.Conclusions:This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin–Stat5–PTHrP axis that is progressively lost in more aggressive tumors.

Purpose: To investigate expression, regulation, potential role and targets of miR-195 and miR-497 in breast cancer.Experimental Design: The expression patterns of miR-195 and miR-497 were initially examined in breast cancer tissues and cell lines by Northern blotting and quantitative real-time PCR. Combined bisulfite restriction analysis and bisulfite sequencing were carried out to study the DNA methylation status of miR-195 and miR-497 genes. Breast cancer cells stably expressing miR-195 and miR-497 were established to study their role and targets. Finally, normal, fibroadenoma and breast cancer tissues were employed to analyze the correlation between miR-195/497 levels and malignant stages of breast tumor tissues.Results: MiR-195 and miR-497 were significantly downregulated in breast cancer. The methylation state of CpG islands upstream of the miR-195/497 gene was found to be responsible for the downregulation of both miRNAs. Forced expression of miR-195 or miR-497 suppressed breast cancer cell proliferation and invasion. Raf-1 and Ccnd1 were identified as novel direct targets of miR-195 and miR-497. miR-195/497 expression levels in clinical specimens were found to be correlated inversely with malignancy of breast cancer.Conclusions: Our data imply that both miR-195 and miR-497 play important inhibitory roles in breast cancer malignancy and may be the potential therapeutic and diagnostic targets. Clin Cancer Res; 17(7); 1722–30. ©2011 AACR.

Background: Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer.Methods: Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium.Results: Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100- to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient = 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors.Conclusions: Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells.Impact: Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy. Cancer Epidemiol Biomarkers Prev; 23(11); 2328–37. ©2014 AACR.

Although patients with advanced ovarian cancer may respond initially to treatment, disease relapse is common, and nearly 50% of patients do not survive beyond five years, indicating an urgent need for improved therapies. To identify new therapeutic targets, we performed single-cell and nuclear RNA-seq data set analyses on 17 human ovarian cancer specimens, revealing the oncostatin M receptor (OSMR) as highly expressed in ovarian cancer cells. Conversely, oncostatin M (OSM), the ligand of OSMR, was highly expressed by tumor-associated macrophages and promoted proliferation and metastasis in cancer cells. Ovarian cancer cell lines and additional patient samples also exhibited elevated levels of OSMR when compared with other cell types in the tumor microenvironment or to normal ovarian tissue samples. OSMR was found to be important for ovarian cancer cell proliferation and migration. Binding of OSM to OSMR caused OSMR–IL6ST dimerization, which is required to produce oncogenic signaling cues for prolonged STAT3 activation. Human monoclonal antibody clones B14 and B21 directed to the extracellular domain of OSMR abrogated OSM-induced OSMR–IL6ST heterodimerization, promoted the internalization and degradation of OSMR, and effectively blocked OSMR-mediated signaling in vitro. Importantly, these antibody clones inhibited the growth of ovarian cancer cells in vitro and in vivo by suppressing oncogenic signaling through OSMR and STAT3 activation. Collectively, this study provides a proof of principle that anti-OSMR antibody can mediate disruption of OSM-induced OSMR–IL6ST dimerization and oncogenic signaling, thus documenting the preclinical therapeutic efficacy of human OSMR antagonist antibodies for immunotherapy in ovarian cancer.Significance:This study uncovers a role for OSMR in promoting ovarian cancer cell proliferation and metastasis by activating STAT3 signaling and demonstrates the preclinical efficacy of antibody-based OSMR targeting for ovarian cancer treatment.

Supplementary Table 1 from Prolactin Inhibits BCL6 Expression in Breast Cancer through a Stat5a-Dependent Mechanism