Publications

Abstract

  • AACR Annual Meeting 2022 Detection of Stemness and EMT in Response to Radiation in Glioblastoma and Ovarian Cancer Cells Aaron Keniston1,2, Alondra Enciso1,2, Yeonkyu Jung2, Ashley Antonissen1,2, Ann Morcos3, Ryan Fuller2, Antonella Bertucci3, Marcelo Vazquez3, Juli Unternaehrer2 1 Department of Biology, California State University San Bernardino, San Bernardino, California 2 Division of Biochemistry, Basic Sciences, Loma Linda University, Loma Linda, California 3 Loma Linda University School of Medicine, Radiation Medicine, Loma Linda, CA   Tumor recurrence and metastasis contribute to the poor prognosis of ovarian cancer (OC) and glioblastoma multiforme (GBM). The aggressiveness of GBM and OC during recurrence creates a need to identify potential therapeutic targets. The cancer stem cell (CSC)-like phenotype and epithelial mesenchymal transition (EMT) have been shown to contribute to cancer aggressiveness, which makes CSCs and EMT viable targets for therapeutic intervention. EMT is a normal process during development and wound healing, but its reactivation in cancer cells contributes to metastasis. To detect CSC phenotypes in live cells without the use of antibodies, we are analyzing core stem cell transcription factors in which a SOX2/OCT4 response element, designated as SORE6, regulates a GFP reporter system.  Also, to detect EMT we are using a GFP based reporter regulated by Zeb1 3’ UTR, a common EMT marker. Our aim is to detect changes in stemness and EMT in response to proton and photon ionizing radiation (IR). We hypothesize that proton and photon IR have similar effects on aggressiveness in OC and GBM. To investigate this, 2 GBM cancer cell lines, 1 breast cancer cell line, 1 OC cell line,  and two patient derived samples were exposed to 0, 1, 2, 4 and 8Gy of 250 MeV proton and 6 MeV photon beams. 72 hours post IR exposure, flow cytometry analysis quantified reporter activity. We found an increase in stemness as radiation dosage increased in most cell lines. Results suggest that proton radiation increases stemness more than photon exposure.   (04/2022)
  • SGO Annual Meeting 2022 Exploring the role of acculturation in promoting genetic testing in the  Latinx community  Ioffe Y 1, Hong L 1, Celestin M  2, Soret C 2,  Schwartz Z,  Montgomery S 2, Unternaehrer JJ 3Objectives: Rates of genetic testing in Latinx women have historically been low. We evaluated the factors contributing to Hispanic/Latinx women’s decision to accept and complete germline genetic testing. We assessed whether acculturation levels in members of the community vs. patients at a comprehensive cancer center (CCC) gynecologic oncology practice were associated with differences in attitudes towards genetic testing. In addition, we hypothesized that mental health, education, and comfort with providers would influence decisions leading to genetic testing. Methods: From 2/2020 to 9/2020, a survey was offered concurrently to Latinx female patients at a CCC gynecologic oncology clinic and to members of a Latinx community group unaffiliated with the CCC. Survey focused on demographic, financial, psychosocial and acculturation factors. Validated scales were utilized, including the PHQ-9, GAD-7 and the BASH acculturation scale. Results: Of the 148 surveys collected, 50% were collected in CCC, 50% in community settings; 42% were completed in Spanish and 58% in English. Most participants (54%) were of reproductive age (<50 years old); 22% had no schooling in the US; 50% had some college/vocational education and 66% had low levels of acculturation. Moreover, 29% of the women were unemployed or did not earn an income; 10% were uninsured, 50% had government subsidized insurance and 40% had private insurance. Most (68%) did not carry a diagnosis of cancer.There was no relationship between age and likelihood of genetic testing, nor with having a cancer diagnosis. Participants from the clinic were more likely to consider genetic screening to determine their risk of cancer before having children compared to those recruited from the community (p=0.004). No correlation was found between mental health (depression or anxiety) and genetic testing in the overall group. However, women over age 50 with higher anxiety had higher testing intentions  (rs=0.21, p=0.048). Women with higher acculturation had higher testing intention (rs=0.54, p=0.001). For women with lower acculturation and who were higher educated in the US, knowing that they would pass on the gene would keep them from getting pregnant (rs=-0.26. p=0.03). Women with lower acculturation were more likely to have genetic testing intentions if they were paired with a female (p=0.02), a Hispanic/Latinx (p=0.001), or a Spanish speaking physician (p=0.001).  Conclusions: In our Latinx participants, those with higher acculturation were more likely to consider genetic testing regardless of clinic or community setting.  For women  with lower acculturation, the identity of the person providing the information mattered  (03/2022)
  • SGO Annual Meeting 2022 Education and context matters when it comes to genetic testing among Latinx women Soret C1,  Celestin M1, Hong L2, Schwartz Z2, Montgomery S1, Unternaehrer JJ3, Ioffe YJ2  Objectives:  Latinx women are known to have significantly lower genetic testing rates compared to other populations despite higher risks for some genetic cancers. To assess attitudes and intentions towards genetic germline testing of Latinx patients in a comprehensive cancer center (CCC) clinic and community group settings, we evaluated demographics and other contextual variables as drivers for the decision to obtain genetic testing.    Methods:  From 2/2020 to 9/2020, a survey was offered concurrently to Latinx female patients at a CCC gynecologic oncology practice and to members of a Latinx community group unaffiliated with a cancer care center. The survey focused on demographic, financial, psychosocial and acculturation factors and genetic testing attitudes and intentions for testing.  Validated scales were utilized to conduct the survey, including the PHQ-9 and GAD-7 and the BASH acculturation scale. Results:  148 evaluable patient surveys were collected, with 50/50 split between the clinic and community settings; 61% of patients were born outside the United States.  42% of patients conducted the surveys in Spanish and 58% in English. Mean patient age was 48, standard deviation 15; 68% of patients did not have cancer. 60% of patients were married or living with a partner. 29% had 0-1 children; 25% had 4 or more children. Those with more children had higher religiosity (rs=.29,p=.03) but it did not affect genetic testing intentions. No correlation was found between patient’s age and genetic testing intentions. Among all women, those who had more schooling outside the US were more likely to have positive attitudes about testing (rs=-.32, p=.005). For women with lower acculturation and with higher education in the US, knowing that they would pass on the gene would be a factor in their decision whether to become pregnant (r-.26. p<.03). Women were more likely to have testing intentions if there was a major life event such as xx (p=0.048). Intention to obtain genetic testing positively correlated with social support (r=0.37, p=0.007 and appropriate medical knowledge about genetic testing (r=0.28, p=0.002). Women who thought that their health insurance covered the testing (p<0.001) and that they would receive information about genetic testing from trusted sources (rs=-.32, p=0.005) had more positive attitudes towards genetic testing.  Conclusions:  If motivated (life events, insurance coverage, social support, trusted information), both clinic and community Latinx women were somewhat open to genetic testing.  Higher educational levels (within and outside the US) resulted in participants being more likely to consider testing. In this heterogeneous group of Latinas residing in Southern California, factors associated with willingness to undergo genetic testing were mainly associated with presence of social support , access, education and medical knowledge, while subjects’ age, religiosity, and number of children were not.  (03/2022)
  • PTCOG 2020 Cancer cell aggressiveness caused by photon vs. proton radiation  (09/2020)
  • (PEER REVIEWED) AACR Annual meetingEvgeny Chirshev1,2, Nozomi Hojo1, Antonella Bertucci1, Linda Sanderman1,3, Anthony Nguyen1, Hanmin Wang1, Tise Suzuki1, Emmanuel Brito1,3, Shannalee R. Martinez4, Christine Castañón1,4, Saied Mirshahidi5, Marcelo E. Vazquez6, Pamela Wat2, Kerby C. Oberg2, Yevgeniya J. Ioffe7, and Juli J. Unternaehrer1,4,8,9 Poster presentation: Epithelial/mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with miRNA let-7 levels and predicts tumor growth and metastasis Objective: Patient-derived samples present an advantage over current cell line models of high grade serous ovarian cancer (HGSOC), which are flawed in terms of being reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we characterized a panel of patient-derived cells to determine their epithelial and mesenchymal characteristics, invasiveness, proliferation, stemness, and in vivo growth.   Experimental procedures: Patient-derived xenograft (PDX) models of HGSOC were analyzed in vitro for phenotypic (RNA, let-7 miRNA, and protein expression, flow cytometry) and functional aspects including growth in spheroids, wound healing assays, invasion assays and in vivo (orthotopic PDX) growth characteristics.    Results: Samples fell along the spectrum from epithelial to mesenchymal, and all had hybrid characteristics. Those toward the more epithelial end of the spectrum were most active in self-renewal assays, and grew most robustly in orthotopic xenograft models. Chemoresistance correlated both with the mesenchymal state and with BRCA2 wild type status. Loss of microRNA let-7 (lethal-7) is an important component of the cancer stem cell phenotype, and we observed an inverse association between let-7 expression and the epithelial state. We observed lower levels of let-7, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy in cells with the most epithelial phenotype.     Conclusions: Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: epithelial cells (those with least let-7 expression) were less migratory, but more tumorigenic, than the mesenchymal cells with higher let-7 expression. We conclude that epithelial/mesenchymal state and let-7 expression are valuable predictors of HGSOC proliferation, in vitro self-renewal, and tumor burden in vivo. (06/2020)
  • ISSCR Annual Meeting 2019 MicroRNA let-7 expression and influence on germ layer ?formation in early mouse development? Chirshev E1, Brito E2,3, Oberg K1,4, and Unternaehrer JJ1,5 MicroRNAs (miRNA) are small non-coding RNAs that negatively regulate gene expression by binding to the 3’ UTR of RNAs. Abnormal regulation and expression of miRNAs have been implemented in various diseases including cancer. The let-7 family of miRNAs was discovered in C. elegans to control developmental timing, and is important for differentiation in mammals. In humans, loss of let-7 contributes to carcinogenesis due to an increase in its target oncogenes and stemness factors. Let-7 regulation is complex and poorly understood. Understanding let-7 regulation and function during normal development can assist in understanding its abnormal regulation in cancer.   Let-7 has been shown to be absent from human embryonic stem cells and increase upon differentiation. During early development, let-7 influences differentiation and germ layer specification, favoring ectoderm and mesoderm. We hypothesize that during mouse early development, let-7 levels demonstrate dynamic expression and its influence on germ layer specification is time dependent. We modeled early mouse development via embryoid body (EB) formation from mESCs. We demonstrated that expression of transcription factor Snail can be used to monitor primitive streak and formation of mesoderm and endoderm. Unexpectedly, our results show a drop in let-7 levels upon exit from pluripotency before it increases, correlating with previously published dynamic changes of let-7 targets HMGA2 and Lin-28. In conclusion, micro-RNA let-7 family members demonstrate dynamic expression during early mouse embryonic development and influence germ layer specification.  (06/2019)
  • Gordon Research Conference: Stem Cells and Cancer Let-7 replacement reduces stemness and aggressiveness of patient-derived ovarian cancer cells Evgeny Chirshev, Nozomi Hojo, Anthony Nguyen, Linda Sanderman, Hanmin Wang, Linda Hong, Yevgeniya J. Ioffe, Juli J. Unternaehrer Objectives: Aggressiveness and recurrence of epithelial ovarian cancer has been attributed to cancer stem cells (CSC) within tumors. Deregulation of miRNAs has been linked to cancer progression, and the stem cell state. Current experiments focused on the tumor suppressor miRNA let-7, loss of which in many cancers is associated with decreased survival. We aimed to understand the role of let-7 in the stem cell-like properties (stemness) of ovarian cancer cells derived from patients, and to test strategies for replacing it. Methods: We characterized a panel of high grade serous ovarian cancer (HGSOC) patient-derived cells. Morphological characteristics, RNA (RT-qPCR) and protein (Western blot and flow cytometry) expression, and miRNA expression were used to determine epithelial (E) vs. mesenchymal (M) phenotype. Functional characteristics including doubling time, growth in spheroids, migration (wound healing assay), chemoresistance (MTT assay), and invasiveness (transwell assay) were determined. Let-7 overexpression was by transfection of miRNA mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six week old nude (J:NU) or NSG mice underwent ovarian bursa injections of HGSOC cells. Results: PDX cells were observed to fall along the spectrum between E/M phenotypes, with the hybrid cells containing most stem cell activity. Some PDX cells were more mesenchymal than previously characterized cell lines; these M cells did not display extensive stemness characteristics. Cells with low let-7 were more stem cell-like, more proliferative, and formed ovarian tumors more robustly. Paradoxically, they were less migratory and more sensitive to cisplatin. Increasing let-7 levels disrupted the stem cell phenotype, and resulted in a reduction in migration and invasion. Conclusions: Patient-derived cells exhibited epithelial, hybrid, or mesenchymal phenotypes, and these correlated with varying degrees of stemness and invasiveness. Hybrid cells are most aggressive. Decreased let-7 levels may predict stemness in HGSOC. In PDX derived from HGSOC patients, in contrast to previously described cell lines, stem cell-like characteristics can be dissociated from the invasive/mesenchymal phenotypes. Let-7 overexpression results in attenuation of the aggressive phenotype, and is a promising therapeutic target in ovarian cancer. (03/2019)
  • SGO annual meetingStem cell-like characteristics of patient-derived high grade serous ovarian cancer xenografts are reversed by miRNA let-7 overexpression Evgeny Chirshev, Nozomi Hojo, Linda Sanderman, Hanmin Wang, Linda Hong, Yevgeniya Ioffe, Juli J. Unternaehrer Objectives: Aggressiveness and recurrence of epithelial ovarian cancer has been attributed to cancer stem cells (CSC) within tumors. Deregulation of miRNAs has been linked to cancer progression, and the stem cell state. Current experiments focused on loss of the tumor suppressor miRNA let-7, which in many cancers is associated with decreased survival. We aimed to understand the role of let-7 in the stem cell-like properties (stemness) of ovarian cancer cells derived from patients. Methods: We characterized a panel of high grade serous ovarian cancer (HGSOC) patient-derived cells. Cell surface expression of CSC markers was analyzed by flow cytometry. Expression at the protein level was detected by Western blot, at RNA level by q-RT-PCR. Migration was determined by wound healing assay, chemoresistance by MTT assay. Let-7 overexpression was by transfection of miRNA mimics. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six week old nude (J:NU) mice underwent ovarian bursa injections of luciferized HGSOC cells. Bioluminescence was quantified by IVIS Lumina III and analyzed by Living Image software. Results: We characterized cells from several PDX for their epithelial (E) vs. mesenchymal (M) and stem cell properties. All PDX cells were classified as E/M hybrids. In contrast to previously characterized cell lines, in PDX the M characteristics did not correlate with stemness. Cells with low let-7 were more stem cell-like, and formed ovarian tumors more robustly. Paradoxically, they were less migratory and more sensitive to cisplatin. Increasing let-7 levels disrupted the stem cell phenotype. Conclusions: PDX derived cells all exhibited hybrid E/M phenotypes. Decreased let-7 levels may predict stemness in HGSOC. In PDX derived from HGSOC patients, in contrast to previously described cell lines, stem cell-like characteristics can be dissociated from the invasive/mesenchymal phenotypes.   (03/2019)
  • Keystone Symposium: Cancer Stem Cells: Advances in Biology and Clinical Translation (EK46) The Snail/let-7 axis induces stemness in cancer cells Wang H, Chirshev E, Hojo N, Lombere M, Huisken A, Unternaehrer J ? One big challenge in cancer therapies is to find effective treatments for patients with advanced tumors. Recurrence and metastasis are caused by a high-stemness subpopulation within tumors. The increase of stemness in cancer cells is associated with the loss of miRNA let-7. The mechanisms of let-7 loss in cancer cells are not completely known. However, evidence from reprogramming studies is consistent with a role for the epithelial-mesenchymal transition (EMT) factor Snail in let-7 repression. EMT is a process by which epithelial cells gain migratory properties to become mesenchymal cells. EMT in cancer cells results in metastases and chemoresistance, indicating an increase in stemness. This leads us to investigate the role of Snail in cancer: does it repress Let-7 directly in cancer cells, as suggested by its role in reprogramming? The goal of our study is to investigate the interaction between Snail and let-7 and its role in regulating cancer cells’ differentiation status. Hence, we hypothesize that Snail directly represses let-7, leading to increased stemness in cancer cells. To approach this, we treated breast (MCF-7), pancreatic (PANC-1), and ovarian cancer cells (OVCAR8, OVSAHO) with TGFb or EGF; both growth factors are known to induce EMT. We also overexpressed Snail by using construct pWZL-Snail. Using qPCR, we measured the expression levels of mRNAs including EMT and stemness markers as well as miRNA let-7. The protein level of said markers was further assessed by immunofluorescence and western blot. Luciferase assay and chromatin immunoprecipitation (CHIP) were used to test the repressing action and the direct binding of Snail on let-7. After either EMT induction or Snail overexpression, an increase in pluripotency markers, as well as a decrease in let-7, was observed, indicating the negative association between Snail and let-7. CHIP data shows that Snail binds to the promoter region of let-7. In addition, luciferase assays revealed that co-transfection of Flag-Snail reduced let-7 promoter activity. These data demonstrate that let-7 is a direct target of Snail. In conclusion, our study has shown that Snail directly represses let-7 transcription and causes an increase in stemness in cancer cells. Snail is thus a potential cancer stemness target in cancer cells. (01/2019)
  • (PEER REVIEWED) Gordon Research Conference: Stem Cells and Cancer. Alyse Huisken1, Nozomi Hojo1, Evgeny Chirshev1,Hanmin Wang1, Maricela Gallardo1,2, Hugo Campos1,3, Carlotta Glackin4, Yevgeniya Ioffe1, Juli Unternaehrer-Hamm1.Snail knockdown restores let-7, reverses stemness, and arrests tumor metastasis in ovarian cancer cells and xenografts.  Background: Chemoresistant cancer stem cells (CSC) are largely responsible for the 70% five-year death rate in epithelial ovarian cancer (EOC). The primary cause of this high mortality rate is tumor recurrence and metastasis. Over 70% of diagnosed ovarian cancers will recur and those that do, rarely respond to treatment. In many cancers, a contributor to the CSC phenotype is loss of tumor suppressor miRNA let-7, due to increased expression of its targets, including oncogenes and pluripotency factors. Mechanisms leading to loss of let-7 are not completely understood. Epithelial-mesenchymal transition (EMT) endows cells with stem cell properties, in addition to its well-defined roles in invasiveness. The EMT factor Snail enhances the transition to stemness in somatic cell reprogramming, and binds promoters of let-7 family miRNAs. We explored the connection between Snail expression and stemness in ovarian cancer cells.   Methods: EMT was induced by culture with epithelial growth factor (EGF). In the EOC line OVSAHO, Snail was overexpressed by estrogen receptor fusion. Snail knockdown (KD) was by lentiviral small hairpin (sh)RNA expression and puromycin selection. Gene expression of mesenchymal, epithelial and pluripotency factors and miRNAs was detected by q-RT-PCR. Chromatin immunoprecipitation of Snail in OVSAHO and 293T was followed by qPCR. Let-7i promoter luciferase was co-transfected into OVCAR8 and 293T cell lines with and without Flag-Snail, and followed by luciferase assays for detection of bioluminescence. Migratory ability was tested in the scratch assay; MTT assays were used to determine drug resistance. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six week old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized EOC cells (OVCAR8 vs PDX) with control vs. Snail KD (250K cells). Bioluminescence was quantified by IVIS Lumina III over 21-50 days and analyzed by Living Image software.   Results: EOC lines were screened on the basis of epithelial and mesenchymal marker expression and morphology, and OVSAHO, COV318, and OVCAR8 were selected for further study in increasing order of mesenchymal characteristics. Snail bound promotors of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7a-3, let-7a-1/f-1/d, and let-7i transcription by Snail. Cells in which Snail was knocked down (KD) expressed less Nanog, and more let-7. Snail KD cells were significantly less migratory than cells with control KD, and were more sensitive to the chemotherapeutic agent cisplatin. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing control or Snail shRNA. Similarly, in PDX derived from ascites, there was a trend toward smaller primary and metastatic tumors.   Conclusions: Snail represses let-7 in ovarian cancer, resulting in an enhanced cancer stem cell phenotype. Snail knockdown disrupts the cancer stem cell phenotype, resulting in less expression of stemness markers, less invasiveness, and increased sensitivity to chemotherapy in vitro, and reduced tumor burden in vivo. Patient-derived xenografts reproducibly phenocopy EOC. We hypothesize that Snail KD-mediated disruption of the cancer stem cell state reverses chemotherapy resistance and slows disease progression in the patient-derived xenograft model. Snail is thus an important therapeutic target in recurrent, metastatic ovarian cancer. (02/2017)
  • (NON-PEER REVIEWED) Cancer Stem Cells Conference: Alyse Huisken-Hill, MS1, Hanmin Wang, BS1, Yevgeniya Ioffe, MD1, Evgeny Chirshev, BS1, Juli Unternaehrer-Hamm, PhD1.  The epithelial-mesenchymal transcription factor Snail contributes to development of stemness and progression of epithelial ovarian cancer metastasis. Background: Chemoresistant cancer stem cells (CSC) are largely responsible for the 70% five-year death rate in epithelial ovarian cancer (EOC). In many cancers, a contributor to the CSC phenotype is loss of miRNA let-7, due to increased expression of its targets, including oncogenes and pluripotency factors. Mechanisms leading to loss of let-7 are incompletely understood. We present preliminary data showing that the epithelial-mesenchymal transition (EMT) factor Snail represses let-7, placing Snail at the nexus of dedifferentiation via loss of let-7, and invasiveness via EMT.   Methods: In the EOC line OVSAHO, Snail was overexpressed by estrogen receptor fusion, or EMT was induced with epidermal growth factor (EGF). Snail knockdown (KD) was by lentiviral small hairpin (sh)RNA expression and puromycin selection. Gene expression of mesenchymal, epithelial and pluripotency factors and miRNAs was detected by q-RT-PCR. Chromatin immunoprecipitation of Snail in OVSAHO and 293T was followed by qPCR. Let-7i promoter luciferase with and without Snail were co-transfected into OVSAHO and 293T cell lines and followed by luciferase assays for detection of bioluminescence. Patient-derived xenografts (PDX) were established subcutaneously in NOD-SCID-Gamma (NSG) mice. Six week old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized EOC cells (OVCAR8 vs PDX) with control vs. Snail KD (250-500K cells). Bioluminescence was quantified by IVIS Lumina III over 21-50 days and analyzed by Living Image software.   Results: EOC lines were screened on the basis of epithelial and mesenchymal marker expression and morphology, and OVSAHO, OVCAR8 and COV318 were selected for further study in increasing order of mesenchymal characteristics. Levels of Snail expression correlated directly with Nanog, and indirectly with let-7 expression. Snail bound promotors of let-7 in cell lines tested. Luciferase assays demonstrated direct repression of let-7 transcription by Snail. Metastatic tumor burden was significantly reduced in orthotopic xenografts using OVCAR8 cells expressing control or Snail shRNA. Similarly, in PDX derived from ascites, there was a trend toward smaller primary and metastatic tumors.   Conclusions: PDX reproducibly phenocopy EOC. Cell line and patient-derived data supports the relationship between Snail expression, let-7 downregulation, and the induction of CSC. We hypothesize that Snail KD disrupts the cancer stem cell state and slows disease progression.   (09/2016)
  • (PEER REVIEWED) Yevgeniya Ioffe1, Alyse Hill2, Linda Sanderman2, Robert Wagner1, Mazdak Momeni1, Linda Hong1, Mark Genesen1, Juli Unternaehrer-Hamm1.WAGO: Epithelial-Mesenchymal Transcription Factor Snail Contributes to Development of Embryonic Phenotype and Progression of Ovarian Epithelial Cancer Metastases Objectives: Metastasis of epithelial ovarian carcinomas (EOC) involves epithelial-mesenchymal transition (EMT) mediated by transcription factors, such as Snail, linked to stem cell phenotype. Snail binds to the promoter region of tumor suppressor let-7 microRNAs (miRNAs). Low let-7 levels correlate with decreased survival in EOC. We aimed to 1) describe the regulation of stemness as impacted by the Snail-let-7 axis in EOC with the use of cell lines; 2) characterize the impact of Snail knock down (KD) on metastasis progression utilizing orthotopic patient-derived mouse xenograft (PDX) model. Methods: Cell lines: In OVSAHO (EOC cell line), Snail was overexpressed by estrogen receptor fusion, or EMT was induced with epidermal growth factor (EGF). Gene expression of Snail, Slug, E-cadherin (mesenchymal/epithelial factors), Nanog, Oct4, Lin28 (embryonic factors), and let-7 family miRNAs was analyzed by quantitative PCR (qPCR). Let-7i promoter luciferase with and without Snail were co-transfected into OVSAHO and 293T (human embryonic kidney) cell lines and followed by measurements of activity. Orthotopic xenografts: Six week old nude (J:NU) mice, 5-8 per experiment, underwent ovarian bursa injections of luciferized patient-derived EOC cells with control vs. Snail KD (500,000 cells). Bioluminescence was quantified (IVIS Lumina) over 21 days. Results: EOC cell lines: OVSAHO: Increased Snail expression resulted in increased Nanog expression (transcription factor in embryonic stem cells) and decreased let-7 expression. Co-transfection of Snail significantly repressed let-7i promoter luciferase activity in OVSAHO and 293T cell lines (p<0.03; 2-tailed t-test), Figure 1.  Orthotopic xenografts: EOC cell line xenograft and PDX reproducibly phenocopy EOC. Preliminary data demonstrate decreased tumor burden via bioluminescence quantification in Snail KD vs. control in PDX (Figure 1). Additional data is being analyzed. Conclusions: We hypothesize that knocking down EMT transcription factor Snail disrupts cancer stem cell (CSC) state and impedes disease progression. Cell line data supports the relationship between Snail overexpression and induction of CSC. Preliminary PDX data demonstrates decrease in metastatic disease with Snail KD. SiRNA delivered by mesoporous silica nanoparticles (MSN) to breast cancer xenografts has been utilized by our collaborators to shut down EMT. Our model can serve to test this mechanism in EOC. Learning objectives:Learn about epithelial mesenchymal transition and metastases in epithelial ovarian cancer.Learn and discuss potential mechanisms of targeting the downregulation of epithelial mesenchymal transition.Identify potential therapeutic targets leading to decrease in metastases progression. (06/2016)

Scholarly Journals--Published

  • Chirshev E, Suzuki T, Wang H, Nguyen A, Hojo N, Sanderman L, Mirshahidi S, Ioffe YJ, Unternaehrer JJ. Let-7i Reduces Aggressive Phenotype and Induces BRCAness in Ovarian Cancer Cells. Cancers (Basel) 2021 Sep 15;13(18):4617. PMID: 34572843 (09/2021)
  • Hanmin Wang, Evgeny Chirshev, Nozomi Hojo, Tise Suzuki, Antonella Bertucci,1 Michael Pierce,2 Christopher Perry,1 Ruining Wang,3 Jeffrey Zink,3 Carlotta A. Glackin,4 Yevgeniya J. Ioffe,5 and Juli J. Unternaehrer1,6, Cancers (Basel). 2021 Mar; 13(6): 1469.Published online 2021 Mar 23. doi: 10.3390/cancers13061469 The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004805/ Simple Summary When cells undergo epithelial–mesenchymal transition (EMT) they gain characteristics of stem cells. We investigated the mechanism by which the EMT transcription factor SNAI1 induces stem cell features. In these studies, we observed that SNAI1 represses a microRNA that maintains differentiation, let-7. This microRNA is lost in cancer, and its loss correlates with poor prognosis. In breast, pancreatic, and ovarian cancer cell lines the cell stemness in increased by SNAI1 overexpression and reduced by SNAI1 knockdown. We extended the ovarian cancer results to patient-derived cells, and to a mouse xenograft model. In mice, we used nanoparticles to deliver small RNAs (RNAi) targeting SNAI1, resulting in restoration of let-7 levels, inhibition of stemness, and reduced tumor burden. Our studies validate nanoparticle-delivered RNAi targeting SNAI1 as a clinically relevant approach. Abstract We aimed to determine the mechanism of epithelial–mesenchymal transition (EMT)-induced stemness in cancer cells. Cancer relapse and metastasis are caused by rare stem-like cells within tumors. Studies of stem cell reprogramming have linked let-7 repression and acquisition of stemness with the EMT factor, SNAI1. The mechanisms for the loss of let-7 in cancer cells are incompletely understood. In four carcinoma cell lines from breast cancer, pancreatic cancer, and ovarian cancer and in ovarian cancer patient-derived cells, we analyzed stem cell phenotype and tumor growth via mRNA, miRNA, and protein expression, spheroid formation, and growth in patient-derived xenografts. We show that treatment with EMT-promoting growth factors or SNAI1 overexpression increased stemness and reduced let-7 expression, while SNAI1 knockdown reduced stemness and restored let-7 expression. Rescue experiments demonstrate that the pro-stemness effects of SNAI1 are mediated via let-7. In vivo, nanoparticle-delivered siRNA successfully knocked down SNAI1 in orthotopic patient-derived xenografts, accompanied by reduced stemness and increased let-7 expression, and reduced tumor burden. Chromatin immunoprecipitation demonstrated that SNAI1 binds the promoters of various let-7 family members, and luciferase assays revealed that SNAI1 represses let-7 transcription. In conclusion, the SNAI1/let-7 axis is an important component of stemness pathways in cancer cells, and this study provides a rationale for future work examining this axis as a potential target for cancer stem cell-specific therapies. Keywords: epithelial–mesenchymal transition, stem cells, ovarian cancer, transcriptional regulation, miRNA, orthotopic patient-derived xenografts (03/2021)
  • Chirshev E, Hojo N, Bertucci A, Sanderman L, Nguyen A, Wang H, Suzuki T, Brito E, Martinez SR, Castanon C, Mirshahidi S, Vazquez M, Wat P, Oberg KC, Ioffe YJ, Unternaehrer JJ. Epithelial/mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with miRNA let-7 levels and predicts tumor growth and metastasis Mol Oncol. 2020 Jul 11. doi: 10.1002/1878-0261.12762 Patient-derived samples present an advantage over current cell line models of high-grade serous ovarian cancer (HGSOC) that are not always reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we set out to characterize a panel of patientderived cells and determine their epithelial and mesenchymal characteristics. We analyzed RNA and protein expression levels in patient-derived xenograft (PDX) models of HGSOC, and functionally characterized these models using flow cytometry, wound healing assays, invasion assays, and spheroid cultures. Besides in vitro work, we also evaluated the growth characteristics of PDX in vivo (orthotopic PDX). We found that all samples had hybrid characteristics, covering a spectrum from an epithelial-to-mesenchymal state. Samples with a stronger epithelial phenotype were more active in self-renewal assays and more tumorigenic in orthotopic xenograft models as compared to samples with a stronger mesenchymal phenotype, which were more migratory and invasive. Additionally, we observed an inverse association between microRNA let-7 (lethal-7) expression and stemness, consistent with the loss of let-7 being an important component of the cancer stem cell phenotype. We observed that lower let-7 levels were associated with the epithelial state and a lower epithelial mesenchymal transition (EMT) score, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy. Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: Cells with lower let- 7 levels were more tumorigenic, but less migratory, and with a lower EMT score, than those with higher let-7 levels. We conclude that let-7 expression and epithelial/mesenchymal state are valuable predictors of HGSOC proliferation, in vitro self-renewal, and tumor burden in vivo. (07/2020)
  • Hanmin Wang and Juli Unternaehrer, Dev Dyn. 2019 Jan;248(1):10-20. doi: 10.1002/dvdy.24678. Epub 2018 Nov 22. Review. PMID: 30303578“Epithelial-mesenchymal Transition and Cancer Stem Cells: At the Crossroads of Differentiation and Dedifferentiation"   In this review, we explore the connection between epithelial-mesenchymal transition (EMT) and differentiation status. Developmental and cancer-related EMTs are described in this context. The evidence for EMT association with both differentiation and dedifferentiation is presented. We hypothesize that EMT causes transient, partial dedifferentiation that functions to allow new gene expression and new cell types in normal development, while in cancer-associated EMTs the dedifferentiation can give rise to a self-renewing population able to re-form tumors. (11/2018) (link)
  • Rowland LK, Campbell PS, Wooten JV, Mavingire N, McLean L, Zylstra D, Thorne G, Daly D, Boyle K, Unternaehrer J, Brantley EJ. J Cell Biochem. 2019 Apr;120(4):6004-6014. doi: 10.1002/jcb.27887. Epub 2018 Nov 18. PMID:30450577 Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand-mediated triple negative breast cancer cell death. (11/2018) (link)
  • Hojo N, Huisken AL, Wang H, Chirshev E, Kim NS, Nguyen SM, Campos H, Glackin CA, Ioffe YJ, Unternaehrer JJ. Sci Rep. 2018 Jun 7;8(1):8704. doi: 10.1038/s41598-018-27021-z. PMID: 29880891 Snail knockdown reverses stemness and inhibits tumour growth in ovarian cancer.To develop effective therapies for advanced high grade serous ovarian cancer (HGSOC), understanding mechanisms of recurrence and metastasis is necessary. In this study, we define the epithelial/mesenchymal status of cell lines that accurately model HGSOC, and evaluate the therapeutic potential of targeting Snai1 (Snail), a master regulator of the epithelial/mesenchymal transition (EMT) in vitro and in vivo. The ratio of Snail to E-cadherin (S/E index) at RNA and protein levels was correlated with mesenchymal morphology in four cell lines. The cell lines with high S/E index (OVCAR8 and COV318) showed more CSC-like, motile, and chemoresistant phenotypes than those with low S/E index (OVSAHO and Kuramochi). We tested the role of Snail in regulation of malignant phenotypes including stemness, cell motility, and chemotherapy resistance: shRNA-mediated knockdown of Snail reversed these malignant phenotypes. Interestingly, the expression of let-7 tumour suppressor miRNA was upregulated in Snail knockdown cells. Furthermore, knockdown of Snail decreased tumour burden in an orthotopic xenograft mouse model. We conclude that Snail is important in controlling HGSOC malignant phenotypes and suggest that the Snail/Let-7 axis may be an attractive target for HGSOC treatment. (06/2018) (link)
  • Shahin SA, Wang R, Simargi SI, Contreras A, Parra Echavarria L, Qu L, Wen W, Dellinger T, Unternaehrer J, Tamanoi F, Zink JI, Glackin CA. Nanomedicine. 2018 Apr 14;14(4):1381-1394. doi: 10.1016/j.nano.2018.04.008. [Epub ahead of print] PMID: 29665439 Hyaluronic acid conjugated nanoparticle delivery of siRNA against TWIST reduces tumor burden and enhances sensitivity to cisplatin in ovarian cancer. (04/2018) (link)
  • Hao J, Unternaehrer J, Cui X, Cheng N, Oji Y. Stem Cells Int. 2017;2017:7681769. doi: 10.1155/2017/7681769. Epub 2017 Jul 27. No abstract available. PMID: 28819365 Regulation of Stemness in Carcinoma Cells. (07/2017) (link)
  • Hao J, Unternaehrer J, Cui X, Cheng N, & Oji Y. (2017). Regulation of Stemness in Carcinoma Cells. Stem Cells Int, 2017, 7681769. (2017) (link)
  • Ioffe Y J M, Hill A, Hojo N, Wang H, Chirshev E, . . . Unternaehrer-Hamm J. (2017). Epithelial-mesenchymal transcription factor Snail leads to cancer stem cell phenotype via repression of tumor suppressor miRNA let-7: Snail knockdown restricts metastatic progression in epithelial ovarian carcinomas. Gynecol Oncol, 145, 105-105. (2017) (link)
  • Kim NS, Mbongue JC, Nicholas DA, Esebanmen GE, Unternaehrer JJ, Firek AF, Langridge WH. PLoS One. 2016 Feb 16;11(2):e0147509. doi: 10.1371/journal.pone.0147509. eCollection 2016. PMID: 26881431   Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-canonical NF-κB Pathway (02/2016) (link)
  • Journal of Immunology: Dequina A. Nicholas*†,  Christopher Hung*†, Kangling Zhang§, Shane Glasgow*†, Aruni Wilson¶, Jacques Mbongue†#, Julia J. Unternaehrer†, Kimberly J. Payne*||, William H.R. Langridge*†, and Marino De Leon*# Palmitic Acid is a Toll-Like Receptor 4 Ligand that Induces Human Dendritic Cell Secretion of Pro-Inflammatory Cytokines (08/2015) (link)
  • Unternaehrer JJ, Zhao R, Kim K, Cesana M, Powers J, Ratanasirintrawoot S, Onder T, Shibue T, Weinberg R, Daley GQ. The epithelial-mesenchymal transition factor SNAI1 paradoxically enhances reprogramming. Stem Cell Reports. 2014 Nov 11;3(5):691-8. doi: 10.1016/j.stemcr.2014.09.008. Epub 2014 Oct 11. Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) entails a mesenchymal to epithelial transition (MET). While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD) of the epithelial-to-mesenchymal transition (EMT) factor SNAI1 (SNAIL) paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency. (11/2014) (link)

Scholarly Journals--Submitted

  • Wang H, Chirshev E, Hojo N, Suzuki T, Bertucci A, Pierce M, Perry C, Wang R, Zink J, Glackin CA, Ioffe YJ, Unternaehrer JJ The epithelial-mesenchymal transcription factor SNAI1 1 represses transcription of the tumor suppressor miRNA let-7 in cancer bioRxiv. 2020. doi: 10.1101/2020.11.04.368688. We aimed to determine the mechanism of epithelial-mesenchymal transition (EMT)-induced stemness in cancer cells. Cancer relapse and metastasis are caused by rare stem-like cells within tumors. Studies of stem cell reprogramming have linked let-7 repression and acquisition of stemness with the EMT factor, SNAI1. The mechanisms for the loss of let-7 in cancer cells are incompletely understood. In four carcinoma cell lines from breast cancer, pancreatic cancer and ovarian cancer and in ovarian cancer patient-derived cells, we analyzed stem cell phenotype and tumor growth via mRNA, miRNA, and protein expression, spheroid formation, and growth in patient-derived xenografts. We show that treatment with EMT-promoting growth factors or SNAI1 overexpression increased stemness and reduced let-7 expression, while SNAI1 knockdown reduced stemness and restored let-7 expression. Rescue experiments demonstrate that the pro-stemness effects of SNAI1 are mediated via let-7. In vivo, nanoparticle-delivered siRNA successfully knocked down SNAI1 in orthotopic patient-derived xenografts, accompanied by reduced stemness and increased let-7 expression, and reduced tumor burden. Chromatin immunoprecipitation demonstrated that SNAI1 binds the promoters of various let-7 family members, and luciferase assays revealed that SNAI1 represses let-7 transcription. In conclusion, the SNAI1/let-7 axis is an important component of stemness pathways in cancer cells, and this study provides a rationale for future work examining this axis as a potential target for cancer stem cell-specific therapies. (11/2020)