Targeting Heat Shock Protein 27 and Fatty Acid Oxidation Augments Cisplatin Treatment in Cisplatin-Resistant Ovarian Cancer Cell Lines.

Most ovarian cancer patients develop recurrent cancers which are often resistant to commonly employed chemotherapy agents, such as cisplatin. We have previously shown that the inhibition of heat shock protein 27 (HSP27) or fatty acid oxidation (FAO) sensitizes cisplatin-resistant ovarian cancer cell lines to cisplatin and dual inhibition of both HSP27 and FAO induces substantial cell death in vitro. However, it is unclear how HSP27 and FAO promote cisplatin resistance, and if dual inhibition of both HSP27 and FAO would augment cisplatin treatment in vivo. Here we showed that HSP27 knockdown in two cisplatin-resistant ovarian cancer cell lines (A2780CIS and PEO4) resulted in more ROS production upon cisplatin treatment. HSP27-knockdown cancer cells exhibited decreased levels of reduced glutathione (GSH) and glucose6phosphate dehydrogenase (G6PD), a crucial pentose phosphate pathway enzyme. ROS depletion with the compound N-acetyl cysteine (NAC) attenuated cisplatin-induced upregulation of HSP27, FAO, and markers of apoptosis and ferroptosis in cisplatin-resistant ovarian cancer cell lines. Finally, inhibition of HSP27 and FAO with ivermectin and perhexiline enhanced the cytotoxic effect of cisplatin in A2780CIS xenograft tumors in vivo. Our results suggest that two different cisplatin-resistant ovarian cancer cell lines upregulate HSP27 and FAO to deplete cisplatin-induced ROS to attenuate cisplatin's cytotoxic effect.

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Cancer-Associated Fibroblasts: Mechanisms of Tumor Progression and Novel Therapeutic Targets.

Cancer-associated fibroblasts (CAFs) are a heterogenous population of stromal cells found in solid malignancies that coexist with the growing tumor mass and other immune/nonimmune cellular elements. In certain neoplasms (e.g., desmoplastic tumors), CAFs are the prominent mesenchymal cell type in the tumor microenvironment, where their presence and abundance signal a poor prognosis in multiple cancers. CAFs play a major role in the progression of various malignancies by remodeling the supporting stromal matrix into a dense, fibrotic structure while secreting factors that lead to the acquisition of cancer stem-like characteristics and promoting tumor cell survival, reduced sensitivity to chemotherapeutics, aggressive growth and metastasis. Tumors with high stromal fibrotic signatures are more likely to be associated with drug resistance and eventual relapse. Clarifying the molecular basis for such multidirectional crosstalk among the various normal and neoplastic cell types present in the tumor microenvironment may yield novel targets and new opportunities for therapeutic intervention. This review highlights the most recent concepts regarding the complexity of CAF biology including CAF heterogeneity, functionality in drug resistance, contribution to a progressively fibrotic tumor stroma, the involved signaling pathways and the participating genes.

Heat Shock Protein 27, a Novel Downstream Target of Collagen Type XI alpha 1, Synergizes with Fatty Acid Oxidation to Confer Cisplatin Resistance in Ovarian Cancer Cells.

Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. We have previously reported that COL11A1 activates Src-Akt signaling through the collagen receptors discoidin domain receptor 2 (DDR2) and integrin α1ß1 to confer cisplatin resistance to ovarian cancer cells. To identify the potential signaling molecules downstream of COL11A1 signaling, we performed protein kinase arrays and identified heat shock protein 27 (HSP27) as a potential mediator of COL11A1-induced cisplatin resistance. Through receptor knockdown and inhibitor experiments, we demonstrated that COL11A1 significantly upregulates HSP27 phosphorylation and expression via DDR2/integrin α1ß1 and Src/Akt signaling in ovarian cancer cells. Furthermore, genetic knockdown and pharmacological inhibition of HSP27, via ivermectin treatment, significantly sensitizes ovarian cancer cells cultured on COL11A1 to cisplatin treatment. HSP27 knockdown or inhibition also decreases NFκB activity as well as the expression of inhibitors of apoptosis proteins (IAPs), which are known downstream effector molecules of COL11A1 that promote cisplatin resistance. Interestingly, HSP27 knockdown or inhibition stimulates ovarian cancer cells to upregulate fatty acid oxidation (FAO) for survival and cisplatin resistance, and dual inhibition of HSP27 and FAO synergistically kills ovarian cancer cells that are cultured on COL11A1. Collectively, this study identifies HSP27 as a novel and druggable COL11A1 downstream effector molecule that may be targeted to overcome cisplatin resistance in recurrent ovarian cancer, which often overexpress COL11A1.

Collagen Type XI Alpha 1 (COL11A1): A Novel Biomarker and a Key Player in Cancer.

Collagen type XI alpha 1 (COL11A1), one of the three alpha chains of type XI collagen, is crucial for bone development and collagen fiber assembly. Interestingly, COL11A1 expression is increased in several cancers and high levels of COL11A1 are often associated with poor survival, chemoresistance, and recurrence. This review will discuss the recent discoveries in the biological functions of COL11A1 in cancer. COL11A1 is predominantly expressed and secreted by a subset of cancer-associated fibroblasts, modulating tumor-stroma interaction and mechanical properties of extracellular matrix. COL11A1 also promotes cancer cell migration, metastasis, and therapy resistance by activating pro-survival pathways and modulating tumor metabolic phenotype. Several inhibitors that are currently being tested in clinical trials for cancer or used in clinic for other diseases, can be potentially used to target COL11A1 signaling. Collectively, this review underscores the role of COL11A1 as a promising biomarker and a key player in cancer.

Targeting progesterone signaling prevents metastatic ovarian cancer.

Effective cancer prevention requires the discovery and intervention of a factor critical to cancer development. Here we show that ovarian progesterone is a crucial endogenous factor inducing the development of primary tumors progressing to metastatic ovarian cancer in a mouse model of high-grade serous carcinoma (HGSC), the most common and deadliest ovarian cancer type. Blocking progesterone signaling by the pharmacologic inhibitor mifepristone or by genetic deletion of the progesterone receptor (PR) effectively suppressed HGSC development and its peritoneal metastases. Strikingly, mifepristone treatment profoundly improved mouse survival (∼18 human years). Hence, targeting progesterone/PR signaling could offer an effective chemopreventive strategy, particularly in high-risk populations of women carrying a deleterious mutation in the BRCA gene.

In vivo modeling of metastatic human high-grade serous ovarian cancer in mice.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.

HOXB13 controls cell state through super-enhancers.

Expression of the homeodomain transcription factor HOXB13 has been demonstrated in several malignancies but its role in tumorigenesis remains elusive. We observed high levels of HOXB13 in poorly differentiated pediatric tumors including a highly aggressive childhood neoplasm - malignant rhabdoid tumor. In a xenograft model of rhabdoid tumor, knockout of HOXB13 diminished tumor growth while partial knockdown of HOXB13 promoted differentiation of tumor cells into bone. These results suggest that HOXB13 enhances rhabdoid malignancy by interfering with mesenchymal stem cell differentiation. Consistent with this hypothesis, overexpression of HOXB13 in mesenchymal progenitor cells inhibited adipogenic, myogenic, and osteogenic differentiation. Mechanistically, we demonstrated that HOXB13 binds to super-enhancer regions regulating genes involved in differentiation and tumorigenesis.

Inhibition of collagen XI alpha 1-induced fatty acid oxidation triggers apoptotic cell death in cisplatin-resistant ovarian cancer.

Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. However, the mechanisms underlying how COL11A1 confers cisplatin resistance in ovarian cancer are poorly understood. We identified that fatty acid ß-oxidation (FAO) is upregulated by COL11A1 in ovarian cancer cells and that COL11A1-driven cisplatin resistance can be abrogated by inhibition of FAO. Furthermore, our results demonstrate that COL11A1 also enhances the expression of proteins involved in fatty acid synthesis. Interestingly, COL11A1-induced upregulation of fatty acid synthesis and FAO is modulated by the same signaling molecules. We identified that binding of COL11A1 to its receptors, α1ß1 integrin and discoidin domain receptor 2 (DDR2), activates Src-Akt-AMPK signaling to increase the expression of both fatty acid synthesis and oxidation enzymes, although DDR2 seems to be the predominant receptor. Inhibition of fatty acid synthesis downregulates FAO despite the presence of COL11A1, suggesting that fatty acid synthesis might be a driver of FAO in ovarian cancer cells. Taken together, our results suggest that COL11A1 upregulates fatty acid metabolism in ovarian cancer cells in a DDR2-Src-Akt-AMPK dependent manner. Therefore, we propose that blocking FAO might serve as a promising therapeutic target to treat ovarian cancer, particularly cisplatin-resistant recurrent ovarian cancers which typically express high levels of COL11A1.

The Role of the Extracellular Matrix in Cancer Stemness.

As our understanding of cancer cell biology progresses, it has become clear that tumors are a heterogenous mixture of different cell populations, some of which contain so called "cancer stem cells" (CSCs). Hallmarks of CSCs include self-renewing capability, tumor-initiating capacity and chemoresistance. The extracellular matrix (ECM), a major structural component of the tumor microenvironment, is a highly dynamic structure and increasing evidence suggests that ECM proteins establish a physical and biochemical niche for CSCs. In cancer, abnormal ECM dynamics occur due to disrupted balance between ECM synthesis and secretion and altered expression of matrix-remodeling enzymes. Tumor-derived ECM is biochemically distinct in its composition and is stiffer compared to normal ECM. In this review, we will provide a brief overview of how different components of the ECM modulate CSC properties then discuss how physical, mechanical, and biochemical cues from the ECM drive cancer stemness. Given the fact that current CSC targeting therapies face many challenges, a better understanding of CSC-ECM interactions will be crucial to identify more effective therapeutic strategies to eliminate CSCs.

Inhibitor of apoptosis proteins (IAPs) mediate collagen type XI alpha 1-driven cisplatin resistance in ovarian cancer.

Although, cisplatin resistance is a major challenge in the treatment of ovarian cancer, the precise mechanisms underlying cisplatin resistance are not fully understood. Collagen type XI alpha 1 (COL11A1), a gene encoding a minor fibrillar collagen of the extracellular matrix, is identified as one of the most upregulated genes in cisplatin-resistant ovarian cancer and recurrent ovarian cancer. However, the exact functions of COL11A1 in cisplatin resistance are unknown. Here we demonstrate that COL11A1 binds to integrin α1ß1 and discoidin domain receptor 2 (DDR2) and activates downstream signaling pathways to inhibit cisplatin-induced apoptosis in ovarian cancer cells. Mechanistically, we show that COL11A1 activates Src-PI3K/Akt-NF-kB signaling to induce the expression of three inhibitor apoptosis proteins (IAPs), including XIAP, BIRC2, and BIRC3. Genetic and pharmacological inhibition of XIAP, BIRC2, and BIRC3 is sufficient to restore cisplatin-induced apoptosis in ovarian cancer cells in the presence of COL11A1 in ovarian cancer cells and xenograft mouse models, respectively. We also show that the components of COL11A1- integrin α1ß1/DDR2- Src-PI3K/Akt-NF-kB-IAP signaling pathway serve as poor prognosis markers in ovarian cancer patients. Taken together, our results suggest novel mechanisms by which COL11A1 confers cisplatin resistance in ovarian cancer. Our study also uncovers IAPs as promising therapeutic targets to reduce cisplatin resistance in ovarian cancer, particularly in recurrent ovarian cancer expressing high levels of COL11A1.

Intracellular Delivery of Nanomaterials via an Inertial Microfluidic Cell Hydroporator.

The introduction of nanomaterials into cells is an indispensable process for studies ranging from basic biology to clinical applications. To deliver foreign nanomaterials into living cells, traditionally endocytosis, viral and lipid nanocarriers or electroporation are mainly employed; however, they critically suffer from toxicity, inconsistent delivery, and low throughput and are time-consuming and labor-intensive processes. Here, we present a novel inertial microfluidic cell hydroporator capable of delivering a wide range of nanomaterials to various cell types in a single-step without the aid of carriers or external apparatus. The platform inertially focuses cells into the channel center and guides cells to collide at a T-junction. Controlled compression and shear forces generate transient membrane discontinuities that facilitate passive diffusion of external nanomaterials into the cell cytoplasm while maintaining high cell viability. This hydroporation method shows superior delivery efficiency, is high-throughput, and has high controllability; moreover, its extremely simple and low-cost operation provides a powerful and practical strategy in the applications of cellular imaging, biomanufacturing, cell-based therapies, regenerative medicine, and disease diagnosis.

FOXC2 augments tumor propagation and metastasis in osteosarcoma.

Osteosarcoma is a highly malignant tumor that contains a small subpopulation of tumor-propagating cells (also known as tumor-initiating cells) characterized by drug resistance and high metastatic potential. The molecular mechanism by which tumor-propagating cells promote tumor growth is poorly understood. Here, we report that the transcription factor forkhead box C2 (FOXC2) is frequently expressed in human osteosarcomas and is important in maintaining osteosarcoma cells in a stem-like state. In osteosarcoma cell lines, we show that anoikis conditions stimulate FOXC2 expression. Downregulation of FOXC2 decreases anchorage-independent growth and invasion in vitro and lung metastasis in vivo, while overexpression of FOXC2 increases tumor propagation in vivo. In osteosarcoma cell lines, we demonstrate that high levels of FOXC2 are associated with and required for the expression of osteosarcoma tumor-propagating cell markers. In FOXC2 knockdown cell lines, we show that CXCR4, a downstream target of FOXC2, can restore osteosarcoma cell invasiveness and metastasis to the lung.

A COL11A1-correlated pan-cancer gene signature of activated fibroblasts for the prioritization of therapeutic targets.

Although cancer-associated fibroblasts (CAFs) are viewed as a promising therapeutic target, the design of rational therapy has been hampered by two key obstacles. First, attempts to ablate CAFs have resulted in significant toxicity because currently used biomarkers cannot effectively distinguish activated CAFs from non-cancer associated fibroblasts and mesenchymal progenitor cells. Second, it is unclear whether CAFs in different organs have different molecular and functional properties that necessitate organ-specific therapeutic designs. Our analyses uncovered COL11A1 as a highly specific biomarker of activated CAFs. Using COL11A1 as a 'seed', we identified co-expressed genes in 13 types of primary carcinoma in The Cancer Genome Atlas. We demonstrated that a molecular signature of activated CAFs is conserved in epithelial cancers regardless of organ site and transforming events within cancer cells, suggesting that targeting fibroblast activation should be effective in multiple cancers. We prioritized several potential pan-cancer therapeutic targets that are likely to have high specificity for activated CAFs and minimal toxicity in normal tissues.

Sphingosine kinase 1 is required for TGF-ß mediated fibroblastto- myofibroblast differentiation in ovarian cancer.

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-ß-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-ß signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

ADAM12 is a prognostic factor associated with an aggressive molecular subtype of high-grade serous ovarian carcinoma.

ADAM metallopeptidase domain 12 (ADAM12) is a promising biomarker because of its low expression in normal tissues and high expression in a variety of human cancers. However, ADAM12 levels in ovarian cancer have not been well characterized. We previously identified ADAM12 as one of the signature genes associated with poor survival in high-grade serous ovarian carcinoma (HGSOC). Here, we sought to determine if high levels of the ADAM12 protein and/or messenger RNA (mRNA) are associated with clinical variables in HGSOC. We show that high protein levels of ADAM12 in banked preoperative sera are associated with shorter progression-free and overall survival. Tumor levels of ADAM12 mRNA were also associated with shorter progression-free and overall survival as well as with lymphatic and vascular invasion, and residual tumor volume following cytoreductive surgery. The majority of genes co-expressed with ADAM12 in HGSOC were transforming growth factor (TGF)ß signaling targets that function in collagen remodeling and cell-matrix adhesion. In tumor sections, the ADAM12 protein and mRNA were expressed in epithelial cancer cells and surrounding stromal cells. In vitro data showed that ADAM12 mRNA levels can be increased by TGFß signaling and direct contact between epithelial and stromal cells. High tumor levels of ADAM12 mRNA were characteristic of the mesenchymal/desmoplastic molecular subtype of HGSOC, which is known to have the poorest prognosis. Thus, ADAM12 may be a useful biomarker of aggressive ovarian cancer for which standard treatment is not effective.

Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC.

Differential expression of argininosuccinate synthetase in serous and non-serous ovarian carcinomas.

The current standard of care for epithelial ovarian cancer does not discriminate between different histologic subtypes (serous, clear cell, endometrioid and mucinous) despite the knowledge that ovarian carcinoma subtypes do not respond uniformly to conventional platinum/taxane-based chemotherapy. Exploiting addictions and vulnerabilities in cancers with distinguishable molecular features presents an opportunity to develop individualized therapies that may be more effective than the current 'one size fits all' approach. One such opportunity is arginine depletion therapy with pegylated arginine deiminase, which has shown promise in several cancer types that exhibit low levels of argininosuccinate synthetase including hepatocellular and prostate carcinoma and melanoma. Based on the high levels of argininosuccinate synthetase previously observed in ovarian cancers, these tumours have been considered unlikely candidates for arginine depletion therapy. However, argininosuccinate synthetase levels have not been evaluated in the individual histologic subtypes of ovarian carcinoma. The current study is the first to examine the expression of argininosuccinate synthetase at the mRNA and protein levels in large cohorts of primary and recurrent ovarian carcinomas and ovarian cancer cell lines. We show that the normal fallopian tube fimbria and the majority of primary high-grade and low-grade serous ovarian carcinomas express high levels of argininosuccinate synthetase, which tend to further increase in recurrent tumours. In contrast to the serous subtype, non-serous ovarian carcinoma subtypes (clear cell, endometrioid and mucinous) frequently lack detectable argininosuccinate synthetase expression. The in vitro sensitivity of ovarian cancer cell lines to arginine depletion with pegylated arginine deiminase was inversely correlated with argininosuccinate synthetase expression. Our data suggest that the majority of serous ovarian carcinomas are not susceptible to therapeutic intervention with arginine deiminase while a subset of non-serous ovarian carcinoma subtypes are auxotrophic for arginine and should be considered for clinical trials with pegylated arginine deiminase.

Effects of the loss of conjunctival Muc16 on corneal epithelium and stroma in mice.

PURPOSE: To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. METHODS: We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. RESULTS: The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. CONCLUSIONS: The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome.