Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

Genomic Integrity Safeguards Self-Renewal in Embryonic Stem Cells.

A multitude of signals are coordinated to maintain self-renewal in embryonic stem cells (ESCs). To unravel the essential internal and external signals required for sustaining the ESC state, we expand upon a set of ESC pluripotency-associated phosphoregulators (PRs) identified previously by short hairpin RNA (shRNA) screening. In addition to the previously described Aurka, we identify 4 additional PRs (Bub1b, Chek1, Ppm1g, and Ppp2r1b) whose depletion compromises self-renewal and leads to consequent differentiation. Global gene expression profiling and computational analyses reveal that knockdown of the 5 PRs leads to DNA damage/genome instability, activating p53 and culminating in ESC differentiation. Similarly, depletion of genome integrity-associated genes involved in DNA replication and checkpoint, mRNA processing, and Charcot-Marie-Tooth disease lead to compromise of ESC self-renewal via an increase in p53 activity. Our studies demonstrate an essential link between genomic integrity and developmental cell fate regulation in ESCs.

Generation of a heterozygous p53 R249S mutant human embryonic stem cell line by TALEN-mediated genome editing.

As one of the most essential genome guardians, p53 and its mutants have been suggested associated with many types of cancers. Many p53 mutants function induce unique phenotypes, including carcinogenesis, metastasis, and drug resistance. The p53(R249S) mutation is the most prevalent and specific mutation associated with liver cancer development. Here, we demonstrate the generation of a heterozygous p53(R249S) mutation in the H9 human embryonic stem cell line using TALEN-mediated genome editing. The generated cell line maintains a normal karyotype, a pluripotent state and the in vivo capacity to develop a teratoma containing all three germ layer tissues.

Oncogenic role of SFRP2 in p53-mutant osteosarcoma development via autocrine and paracrine mechanism.

Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a ß-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.

Generation of an induced pluripotent stem cell line from an individual with a heterozygous RECQL4 mutation.

The DNA helicase RECQL4 is known for its roles in DNA replication and repair. RECQL4 mutations cause several genetic disorders including Rothmund-Thomson syndrome (RTS), characterized by developmental defects and predisposition to osteosarcoma. Here we reprogrammed fibroblasts with a heterozygous RECQL4 mutation (c.1878 + 32_1878 + 55del24) to induced pluripotent stem cells (iPSCs). These iPSCs are pluripotent and are able to be differentiated into all three germ layers, providing a novel tool to further interrogate the role of RECQL4 DNA helicase in vitro.

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Establishment of a human embryonic stem cell line with homozygous TP53 R248W mutant by TALEN mediated gene editing.

Genetic mutations in TP53 contribute to multiple human cancers. Here we report the generation of a H1-p53(R248W/R248W) human embryonic stem cell line harboring a homozygous TP53 R248W mutation created by TALEN-mediated precise gene editing. The H1-p53(R248W/R248W) cell line maintains a normal karyotype, robust pluripotency gene expression, and the potential to differentiate to the three germ layers.

A homozygous p53 R282W mutant human embryonic stem cell line generated using TALEN-mediated precise gene editing.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancers. Many hot-spot mutations of TP53 confer novel functions not found in wild-type p53 and contribute to tumor development and progression. We report on the generation of a H1 human embryonic stem cell line carrying a homozygous TP53 R282W mutation using TALEN-mediated genome editing. The generated cell line demonstrates normal karyotype, maintains a pluripotent state, and is capable of generating a teratoma in vivo containing tissues from all three germ layers.

Generation of human embryonic stem cell line with heterozygous RB1 deletion by CRIPSR/Cas9 nickase.

The Retinoblastoma 1 (RB1) tumor suppressor, a member of the Retinoblastoma gene family, functions as a pocket protein for the functional binding of E2F transcription factors. About 1/3 of retinoblastoma patients harbor a germline RB1 mutation or deletion, leading to the development of retinoblastoma. Here, we demonstrate generation of a heterozygous deletion of the RB1 gene in the H1 human embryonic stem cell line using CRISPR/Cas9 nickase genome editing. The RB1 heterozygous knockout H1 cell line shows a normal karyotype, maintains a pluripotent state, and is capable of differentiation to the three germline layers.

Differential expression and clinical significance of epithelial-mesenchymal transition markers among different histological types of triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease closely associated with epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the role of EMT in metaplastic carcinoma. Methods: E-cadherin, Slug, Twist and Vimentin levels were detected by immunohistochemistry in 167 TNBC tumors, including 145 invasive carcinomas of no special type (ICONSTs), 14 spindle cell carcinomas (SpCCs) and 8 matrix-producing carcinomas (MPCs). Results: Nuclear Slug and Twist were more frequently detected in SpCC and MPC tumors than that in ICONST tumors (p<0.001). The rate of E-cadherin loss was much lower in the ICONST tumors than that in the SpCC and MPC tumors (p<0.001). Vimentin was expressed in all SpCC and MPC tumors. Furthermore, nuclear expression of Slug and Twist was positively associated with the cytoplasmic localization of Vimentin (p<0.001) and was inversely associated with membranous staining of E-cadherin (p<0.001). These trends were more apparent in the SpCC and MPC tumors than in the ICONST tumors. Follow-up data were available for 151 patients. The follow-up times ranged from 1 month to 11 years (mean: 74 m; median: 21 m). The median progression-free survival and overall survival times were 24 months (mean: 32 months) and 22 months (mean: 35 months), respectively. Tumor size, TNM stage and E-cadherin were found to be independent prognostic factors of TNBC. Conclusions: EMT may play an important role in TNBC, especially in MPC and SpCC. Further researches are needed to confirm this finding. The results of this study may facilitate the future development of targeted therapies based on alterations in the EMT and stem cell markers.

Li-Fraumeni Syndrome Disease Model: A Platform to Develop Precision Cancer Therapy Targeting Oncogenic p53.

Li-Fraumeni syndrome (LFS) is a rare hereditary autosomal dominant cancer disorder. Germline mutations in TP53, the gene encoding p53, are responsible for most cases of LFS. TP53 is also the most commonly mutated gene in human cancers. Because inhibition of mutant p53 is considered to be a promising therapeutic strategy to treat these diseases, LFS provides a perfect genetic model to study p53 mutation-associated malignancies as well as to screen potential compounds targeting oncogenic p53. In this review we briefly summarize the biology of LFS and current understanding of the oncogenic functions of mutant p53 in cancer development. We discuss the strengths and limitations of current LFS disease models, and touch on existing compounds targeting oncogenic p53 and in vitro clinical trials to develop new ones. Finally, we discuss how recently developed methodologies can be integrated into the LFS induced pluripotent stem cell (iPSC) platform to develop precision cancer therapy.

Modeling Cancer with Pluripotent Stem Cells.

The elucidation of cancer pathogenesis has been hindered by limited access to patient samples, tumor heterogeneity and the lack of reliable model organisms. Characterized by their ability to self-renew indefinitely and differentiate into all cell lineages of an organism, pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide a powerful and unlimited source to generate differentiated cells that can be used to study disease biology, facilitate drug discovery and development, and provide key insights for developing personalized therapies. This article reviews the recent developments and technologies converting PSCs into clinically relevant model systems for cancer research.

Genomic Landscape of Colorectal Mucosa and Adenomas.

The molecular basis of the adenoma-to-carcinoma transition has been deduced using comparative analysis of genetic alterations observed through the sequential steps of intestinal carcinogenesis. However, comprehensive genomic analyses of adenomas and at-risk mucosa are still lacking. Therefore, our aim was to characterize the genomic landscape of colonic at-risk mucosa and adenomas. We analyzed the mutation profile and copy number changes of 25 adenomas and adjacent mucosa from 12 familial adenomatous polyposis patients using whole-exome sequencing and validated allelic imbalances (AI) in 37 adenomas using SNP arrays. We assessed for evidence of clonality and performed estimations on the proportions of driver and passenger mutations using a systems biology approach. Adenomas had lower mutational rates than did colorectal cancers and showed recurrent alterations in known cancer driver genes (APC, KRAS, FBXW7, TCF7L2) and AIs in chromosomes 5, 7, and 13. Moreover, 80% of adenomas had somatic alterations in WNT pathway genes. Adenomas displayed evidence of multiclonality similar to stage I carcinomas. Strong correlations between mutational rate and patient age were observed in at-risk mucosa and adenomas. Our data indicate that at least 23% of somatic mutations are present in at-risk mucosa prior to adenoma initiation. The genomic profiles of at-risk mucosa and adenomas illustrate the evolution from normal tissue to carcinoma via greater resolution of molecular changes at the inflection point of premalignant lesions. Furthermore, substantial genomic variation exists in at-risk mucosa before adenoma formation, and deregulation of the WNT pathway is required to foster carcinogenesis. Cancer Prev Res; 9(6); 417-27. ©2016 AACR.

Male breast carcinoma: a clinicopathological and immunohistochemical characterization study.

Male breast carcinoma is a relatively rare disease. This study retrospectively investigated the clinicopathological features of 73 cases of male breast carcinoma in Chinese population, and classified the molecular subtype based on surrogate immunohistochemical definitions. The expression of GCDFP15, MGB, AR and FOXP1 were evaluated. Invasive carcinoma of no special type was the most common histological type in the study group (71.2%, 52/73). The luminal A and B subtypes were the major types of male breast carcinoma (60.9%, 34.8% respectively). AR and FOXP1 are expressed in 84.2% (48/57) and 71.9% (41/57) of the studied cases. Carcinoma of the luminal A subtype expressed GCDFP15 (73.5%, 25/34) and MGB (58.8%, 20/34) more frequently than cases of the luminal B subtypes (34.8%, 8/23 and 43.5%, 10/23, respectively; P = 0.004, P = 0.255, respectively). In conclusion, invasive carcinoma of no special type was the most common histological type in male breast carcinoma among Chinese population. Our study revealed that the luminal A and B subtypes were the major types of male breast carcinoma. AR and FOXP1 are highly expressed in male breast cancer. The luminal A subtype tends to express GCDFP15 and MGB more frequently than the luminal B subtype.

CDK11p58 inhibits ERα-positive breast cancer invasion by targeting integrin ß3 via the repression of ERα signaling.

BACKGROUND: CDK11(p58), a Ser/Thr kinase that belongs to the cell division cycle 2-like 1 (CDC2L1) subfamily, is associated with cell cycle progression, tumorigenesis and apoptotic signaling. CDK11(p58) is also involved in the regulation of steroid receptors, such as androgen and estrogen receptors. We previously found that CDK11(p58) was abnormally expressed in prostate cancer. However, its role in breast cancer remains unclear. METHODS: CDK11(p58) expression was evaluated by immunohistochemical staining in a tissue array. A Transwell assay was used to detect invasion and metastasis in breast cancer cells. The TaqMan® Metastasis Gene Expression Assay was used to search for potential downstream factors in the CDK11(p58) signaling pathway. qRT-PCR was used to evaluate mRNA levels, and the dual luciferase array was used to analyze promoter activity. Western blotting was used to detect the protein level. RESULTS: CDK11(p58) expression was negatively correlated with node status (P = 0.012), relapse status (P = 0.002) and metastasis status (P = 0.023). Kaplan-Meier survival curves indicated that the disease-free survival (DFS) was significantly poor in breast cancer patients with low CDK11 expression. Interestingly, using the breast cancer cell lines ZR-75-30 and MDA-MB-231, we found that CDK11(p58) was capable of repressing the migration and invasion of ERα-positive breast cancer cells, but not ERα-negative breast cancer cells, in a kinase-dependent manner. Gene expression assays demonstrated that integrin ß3 mRNA was dramatically repressed by CDK11(p58), and luciferase results confirmed that the integrin ß3 promoter was inhibited by CDK11(p58) through ERα repression. The expression of integrin ß3 was highly related to ERα signaling; ERα overexpression stimulated integrin ß3 expression, whereas siRNA-mediated knockdown of ERα attenuated integrin ß3 expression. CONCLUSIONS: These data indicate that CDK11(p58) is an anti-metastatic gene in ERα-positive breast cancer and that the regulation of integrin ß3 by CDK11(p58) via the repression of ERα signaling may constitute part of a signaling pathway underlying breast cancer invasion.

Invasive breast carcinomas of no special type with osteoclast-like giant cells frequently have a luminal phenotype.

Invasive carcinoma of no special type (NST) with osteoclast-like giant cells (OGCs) represents a unique type of breast neoplasm, characterized by the presence of multinucleated OGCs and a vascularized, hemorrhagic stroma. Because of its rarity, the literature regarding this tumor remains limited and a detailed immunophenotype of this tumor has not been established as yet. We report a clinicopathological and immunohistochemical study of 42 patients with invasive carcinoma NST with OGCs. Macroscopically, these tumors presented as a well-delimited red-brown mass. A remarkable feature of the tumor was the presence of OGCs in the fibroblastic or hemorrhagic vascular stroma, as well as in the adjacent tumor nests or glandular lumina. The number of OGCs varied from 8 to 105 per 10 high-power fields with an average of 48. The tumors were well to moderately differentiated. Cribriform architecture was observed in 27 tumors (63 %). All of the 36 available tumors were of luminal phenotype, according to the Ki67 labeling index 89 % luminal A and 11 % luminal B. With a mean follow-up time of 46.4 months, lung metastasis was found in 2 patients (5 %) at 7 and 11 years after the operation, respectively. None of the other cases had presented with evidence of recurrence or metastasis. To the best of our knowledge, this is the largest reported series of invasive carcinoma NST with OGCs as yet. Our study revealed that invasive carcinoma NST with OGCs exhibit a luminal phenotype with luminal A subtype as the major group.

The residual tumor autophagy marker LC3B serves as a prognostic marker in local advanced breast cancer after neoadjuvant chemotherapy.

PURPOSE: This study sought to investigate the prognostic value of the autophagy marker microtubule-associated protein chain 3B (LC3B) in patients with residual tumors after neoadjuvant chemotherapy (NCT) for locally advanced breast cancer (LABC). PATIENTS AND METHODS: The expression of LC3B in residual breast cancer cells was assessed by immunohistochemistry in surgical specimens from 229 patients diagnosed with histologically proven invasive breast cancer. All patients underwent NCT followed by mastectomy and were considered nonpathologic complete responders (non-pCR) after a pathologic evaluation. The prognostic value of various clinicopathologic factors was evaluated. RESULTS: The LC3B density was similar between the peripheral and central area of the tumors (P = 0.328) but was significantly lower in the extratumoral area (P < 0.001 and P < 0.001, respectively). Furthermore, LC3B density, which correlated with Beclin-1 expression, Ki-67 index, and breast cancer subtype, served as an independent prognostic factor for both relapse-free survival (RFS; P = 0.012) and overall survival (OS; P = 0.008); the prognostic value of LC3B was most significant in triple-negative patients. Using a combination of LC3B expression and the status of residual involved lymph nodes, the patients were classified into four groups with different risks of relapse and death (P < 0.001 for RFS and P = 0.003 for OS). CONCLUSION: LC3B can be used as a prognostic marker in patients with non-pCR after NCT for breast cancer, which highlights the importance of autophagy in the biologic behavior of chemoresistant cancer cells. Furthermore, evaluating and targeting autophagy in the neoadjuvant setting may help prevent disease relapse in patients with non-pCR.

Rhabdomyosarcoma of the breast: a clinicopathologic study and review of the literature.

BACKGROUND: Rhabdomyosarcoma (RMS) is an uncommon malignancy of the breast. The aim of this study was to summarize its clinicopathologic features and biological behavior. METHODS: Five primary or secondary breast RMSs were collected. Their clinicopathological characteristics and all published literature about breast RMS were reviewed. Immunohistochemical study of desmin, myogenic differentiation 1 (MyoD1), myogenin, leukocyte common antigen (LCA), vimentin, cytokeratin (AE1/AE3), E-cadherin, neuron specific enolase (NSE), CD99, chorioallantoic membrane 5.2 (CAM5.2) and epithelial membrane antigen (EMA) expression were performed. RESULTS: The five patients were all female with ages ranging from 16 to 46 years old (mean, 30 years). Three were metastatic breast RMSs, two embryonal and one solid variant alveolar, with the primary tumor sites the right labium majus, left nasal meatus and nasopharynx, respectively. The other two, one embryonal and one alveolar, were primaries. Grossly, the surgical specimens revealed round or oval, well-demarcated but nonencapsulated masses. Their cut surfaces consisted of homogeneous grayish yellow or white tissue. Microscopically, most tumor cells were poorly differentiated small round, oval or small polygons with eosinophilic cytoplasm. All cases were positive for vimentin, desmin, MyoD1 and myogenin. One embryonal RMS also had a few cells with perinuclear staining of AE1/AE3. The other markers were negative. CONCLUSIONS: Although primary or metastatic RMS in breast was almost confined to young adolescent females, our cases suggested that it can also happen to the middle-aged women. Embryonal RMS has a certain metastatic potential. MyoD1 and myogenin are two useful markers when making differential diagnosis. Axillary lymph node status and age may play a role in the prognosis of primary breast RMS patients.

Obesity or overweight is associated with worse pathological response to neoadjuvant chemotherapy among Chinese women with breast cancer.

BACKGROUND: To evaluate the relationship between body mass index (BMI) and response to neoadjuvant chemotherapy (NCT) for breast cancer among Chinese women. PATIENTS AND METHODS: A total of 307 eligible patients were assigned to receive four cycles of paclitaxel and carboplatin before standard surgery for breast cancer from 2007 to 2011 at Shanghai Cancer Hospital. The patients were categorized as obese, overweight, normal weight, or underweight based on BMI according to World Health Organization (WHO) criteria. Pathological complete response (pCR) was defined as no invasive cancer in the breast or axillary tissue. A logistic regression and the Chi-squared test were used for detecting the predictors of pCR and determining the relationship between BMI category and pCR rate in the subgroup analysis with respect to other variables. RESULTS: Categorical BMI, estrogen receptor (ER), and progesterone receptor (PR) status were independent predictors of pCR according to the multivariate analysis. Patients with BMI≥25 were less likely to achieve a pCR to NCT compared with patients with BMI<25 (Odds ratio: 0.454, p = 0.033, multivariate analysis). In the subgroup analysis, the predictive value of BMI for pCR to NCT was significantly shown in post-menopausal patients (p = 0.004) and hormonal receptor status-negative patients (p = 0.038). The incidence of treatment-induced toxicity was similar among the different BMI categories. CONCLUSION: Higher BMI was associated with worse pCR to NCT. Further approaches to investigating the mechanism of this influence of BMI on treatment response and a more appropriate schedule for calculating NCT dose for high-BMI-patients should be considered.

Prognostic value of a positive-to-negative change in hormone receptor status after neoadjuvant chemotherapy in patients with hormone receptor-positive breast cancer.

BACKGROUND: To investigate the prognostic value of positive-to-negative changes in hormone receptor (HR) status after neoadjuvant chemotherapy (NCT) in patients with HR-positive breast cancer. METHODS: Data from 224 stage II-III breast cancer patients with positive HR status before NCT who had residual disease in the breast after NCT were collected. HR status of the residual tumors was retested after NCT. A survival analysis was performed in 214 patients with adjuvant endocrine therapy regardless of post-NCT HR status. The survival analysis also examined other clinical and pathologic variables. RESULTS: In total, 15.2 % of patients had a positive-to-negative change in HR status after NCT, and this change was observed more frequently in HER-2-positive tumors than HER-2-negative tumors (P = 0.001). In 214 patients who had been treated with adjuvant endocrine therapy regardless of post-NCT HR status, the alteration in HR status was an independent factor for the prediction of a poorer disease-free survival (P = 0.026) and overall survival (P < 0.001) in the adjuvant endocrine therapy patients. The 5-year disease-free survival and overall survival rates were 43.5 % and 59.8 %, respectively, in patients with HR status conversion and 67.8 % and 82.5 %, respectively, in patients whose HR status remained positive (log rank test P = 0.003 and P = 0.001). CONCLUSIONS: The switch of HR status after NCT is remarkable for HR-positive tumors. An HR-negative switch may identify patients who would benefit from alternative systemic therapies.