Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment.

Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.

Enniatin A inhibits the chaperone Hsp90 and unleashes the immune system against triple-negative breast cancer.

Low response rates and immune-related adverse events limit the remarkable impact of cancer immunotherapy. To improve clinical outcomes, preclinical studies have shown that combining immunotherapies with N-terminal Hsp90 inhibitors resulted in improved efficacy, even though induction of an extensive heat shock response (HSR) and less than optimal dosing of these inhibitors limited their clinical efficacy as monotherapies. We discovered that the natural product Enniatin A (EnnA) targets Hsp90 and destabilizes its client oncoproteins without inducing an HSR. EnnA triggers immunogenic cell death in triple-negative breast cancer (TNBC) syngeneic mouse models and exhibits superior antitumor activity compared to Hsp90 N-terminal inhibitors. EnnA reprograms the tumor microenvironment (TME) to promote CD8+ T cell-dependent antitumor immunity by reducing PD-L1 levels and activating the chemokine receptor CX3CR1 pathway. These findings provide strong evidence for transforming the immunosuppressive TME into a more tumor-hostile milieu by engaging Hsp90 with therapeutic agents involving novel mechanisms of action.

Capsaicin binds the N-terminus of Hsp90, induces lysosomal degradation of Hsp70, and enhances the anti-tumor effects of 17-AAG (Tanespimycin).

Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.

RAD51AP1 Loss Attenuates Colorectal Cancer Stem Cell Renewal and Sensitizes to Chemotherapy.

DNA damage, induced by either chemical carcinogens or environmental pollutants, plays an important role in the initiation of colorectal cancer. DNA repair processes, however, are involved in both protecting against cancer formation, and also contributing to cancer development, by ensuring genomic integrity and promoting the efficient DNA repair in tumor cells, respectively. Although DNA repair pathways have been well exploited in the treatment of breast and ovarian cancers, the role of DNA repair processes and their therapeutic efficacy in colorectal cancer is yet to be appreciably explored. To understand the role of DNA repair, especially homologous recombination (HR), in chemical carcinogen-induced colorectal cancer growth, we unraveled the role of RAD51AP1 (RAD51-associated protein 1), a protein involved in HR, in genotoxic carcinogen (azoxymethane, AOM)-induced colorectal cancer. Although AOM treatment alone significantly increased RAD51AP1 expression, the combination of AOM and dextran sulfate sodium (DSS) treatment dramatically increased by several folds. RAD51AP1 expression is found in mouse colonic crypt and proliferating cells. RAD51AP1 expression is significantly increased in majority of human colorectal cancer tissues, including BRAF/KRAS mutant colorectal cancer, and associated with reduced treatment response and poor prognosis. Rad51ap1-deficient mice were protected against AOM/DSS-induced colorectal cancer. These observations were recapitulated in a genetically engineered mouse model of colorectal cancer (ApcMin /+ ). Furthermore, chemotherapy-resistant colorectal cancer is associated with increased RAD51AP1 expression. This phenomenon is associated with reduced cell proliferation and colorectal cancer stem cell (CRCSC) self-renewal. Overall, our studies provide evidence that RAD51AP1 could be a novel diagnostic marker for colorectal cancer and a potential therapeutic target for colorectal cancer prevention and treatment. IMPLICATIONS: This study provides first in vivo evidence that RAD51AP1 plays a critical role in colorectal cancer growth and drug resistance by regulating CRCSC self-renewal.

Critical immunosuppressive effect of MDSC­derived exosomes in the tumor microenvironment.

Myeloid­derived suppressor cells (MDSCs) are an indispensable component of the tumor microenvironment (TME). Along with the role of MDSC immunosuppression and antitumor immunity, MDSCs facilitate tumor growth, differentiation, and metastasis in several ways that are yet to be explored. Like any other cell type, MDSCs also release a tremendous number of exosomes, or nanovesicles of endosomal origin, that participate in intercellular communications by dispatching biological macromolecules. There have been no investigational studies conducted to characterize the role of MDSC­derived exosomes (MDSC exo) in modulating the TME. In this study, we isolated MDSC exo and demonstrated that they carry a significant level of proteins that play an indispensable role in tumor growth, invasion, angiogenesis, and immunomodulation. We observed a higher yield and more substantial immunosuppressive potential of exosomes isolated from MDSCs in the primary tumor area than those in the spleen or bone marrow. Our in vitro data suggest that MDSC exo are capable of hyper­activating or exhausting CD8 T­cells and induce reactive oxygen species production that elicits activation­induced cell death. We confirmed the depletion of CD8 T­cells in vivo by treating mice with MDSC exo. We also observed a reduction in pro­inflammatory M1­macrophages in the spleen of those animals. Our results indicate that the immunosuppressive and tumor­promoting functions of MDSCs are also implemented by MDSC­derived exosomes which would open up a new avenue of MDSC research and MDSC­targeted therapy.

RAD51AP1 Deficiency Reduces Tumor Growth by Targeting Stem Cell Self-Renewal.

RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor-positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo, limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer. SIGNIFICANCE: This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.

Short-Term Diet Restriction but Not Alternate Day Fasting Prevents Cisplatin-Induced Nephrotoxicity in Mice.

Cisplatin (CP) is one of the most preferred platinum-containing antineoplastic drugs. However, even in nontoxic plasma concentrations, it may cause kidney injury. To be able to increase its effective pharmacological dose, its side effects need to be regarded. Diet restriction (DR) has been demonstrated to improve cellular survival in a number of disorders. In this context, we investigated the role of DR in CP-induced nephrotoxicity (CPN). Besides alternate DR, animals were exposed to DR for 3 days prior or after CP treatment. Here, we observed that both 3 days of DR reverses the nephrotoxic effect of CP, which was associated with improved physiological outcomes, such as serum creatine, blood-urea nitrogen and urea. These treatments significantly increased phosphorylation of survival kinases PI3K/Akt and ERK-1/2 and decreased the level of stress kinase JNK were noted. In addition, the activation level of signal transduction mediator p38 MAPK phosphorylation was higher particularly in both three-day DR groups. Next, animals were fed with carbohydrate-, protein- or fat-enriched diets in the presence of CP. Results indicated that not only fasting but also dietary content itself may play a determinant role in the severity of CPN. Our data suggest that DR is a promising approach to reduce CPN by regulating metabolism and cell signaling pathways.

Correction: The pleiotropic effects of TNFα in breast cancer subtypes is regulated by TNFAIP3/A20.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plasticity and Potency of Mammary Stem Cell Subsets During Mammary Gland Development.

It is now widely believed that mammary epithelial cell plasticity, an important physiological process during the stages of mammary gland development, is exploited by the malignant cells for their successful disease progression. Normal mammary epithelial cells are heterogeneous and organized in hierarchical fashion, in which the mammary stem cells (MaSC) lie at the apex with regenerative capacity as well as plasticity. Despite the fact that the majority of studies supported the existence of multipotent MaSCs giving rise to both basal and luminal lineages, others proposed lineage restricted unipotent MaSCs. Consistent with the notion, the latest research has suggested that although normal MaSC subsets mainly stay in a quiescent state, they differ in their reconstituting ability, spatial localization, and molecular and epigenetic signatures in response to physiological stimuli within the respective microenvironment during the stages of mammary gland development. In this review, we will focus on current research on the biology of normal mammary stem cells with an emphasis on properties of cellular plasticity, self-renewal and quiescence, as well as the role of the microenvironment in regulating these processes. This will include a discussion of normal breast stem cell heterogeneity, stem cell markers, and lineage tracing studies.

Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2-/- mice, an effect mimicked by CD8+ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8+ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression.

The co-chaperone UNC45A is essential for the expression of mitotic kinase NEK7 and tumorigenesis.

Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types.

The pleiotropic effects of TNFα in breast cancer subtypes is regulated by TNFAIP3/A20.

TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.

The EGFR T790M Mutation Is Acquired through AICDA-Mediated Deamination of 5-Methylcytosine following TKI Treatment in Lung Cancer.

: Almost all patients with EGFR-driven lung cancer who are treated with EGFR tyrosine kinase inhibitors (TKI) develop resistance to treatment. A single base (c.2369C>T) transition mutation, EGFR T790M, is the most frequent resistance event after first-generation exposure to EGFR TKIs. Whether T790M mutation is acquired or is selected from a preexisting clone has been a matter of significant debate. In this study, we show that treatment with EGFR TKIs leads to activation of the NFκB pathway, which in turn induces expression of activation-induced cytidine deaminase (AICDA). In turn, AICDA causes deamination of 5-methylcytosine to thymine at position c.2369 to generate the T790M mutation. Pharmacologic inhibition of the NFκB pathway or knockout of AICDA decreased the frequency or prevented the development of T790M mutation, respectively. In addition, patients treated with first-line EGFR TKI displayed increased expression of AICDA and detection of the T790M mutation upon progression. These results identify the mechanism of T790M acquisition and present an opportunity to target the process to delay or prevent it. SIGNIFICANCE: These findings identify the mechanism behind acquisition of a common resistance mutation to TKI treatment in lung cancer.

Thymoquinone prevents cisplatin neurotoxicity in primary DRG neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a substantial, dose-limiting adverse effect that occurs in cancer patients. Cis-dichlorodiamine (II) platinum (CDDP, cisplatin) is a platinum-based chemotherapeutic agent that causes severe acute and chronic peripheral neuropathies in 30% of cancer patients. Thymoquinone (TQ), a leading bioactive constituent of Nigella sativa seeds, has been reported to have antioxidant, anti-inflammatory, anti-neoplastic and neuroprotective properties. Dorsal root ganglia (DRG) include different classes of primary sensory neurons, such as nociceptors, mechanoreceptors, and proprioceptive neurons. Here, we investigated the neuroprotective activity of TQ against cisplatin neurotoxicity in cultured DRG neurons. We prepared neuronal cultures from DRGs of adult mice, pre-treated them with or without varying doses of TQ prior to exposure of cells to cisplatin. The preparations were viewed under the scope before and after the treatment at 24 h, 48 h, and 72 h time points. We analyzed neuronal cell viability and neurite outgrowths, and evaluated morphologic changes of neuronal or non-neuronal cells. TQ significantly increases the ability to extend neurites and neuronal cell viability when compared to the culture conditions which were treated with cisplatin only. Although we provide compelling evidence for the protective activity of TQ against chemotherapy-induced neurotoxicity, further detailed investigations in preclinical settings are warranted for its clinical use.

Thymoquinone protects DRG neurons from axotomy-induced cell death.

OBJECTIVE: Peripheral nerve injury (PNI) is a significant health problem that is linked to sensory, motor, and autonomic deficits. This pathological condition leads to a reduced quality of life in most affected individuals. Schwann cells (SCs) play a crucial role in the repair of PNI. Effective agents that promote SC activation may facilitate and accelerate peripheral nerve repair. Thymoquinone (TQ), a bioactive component of Nigella sativa seeds, has an antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective properties. In the present study, the neuroprotective efficacy of TQ was investigated by using a laser microdissection technique in a mouse PNI model. METHODS: Single cells were isolated from dorsal root ganglions (DRGs) of 6-8-week-old mice, maintained in defined culture conditions and treated with or without TQ at different concentrations. Axons were cut (axotomy) using a controllable laser microbeam to model axonal injury in vitro. Under fluorescence microscopy, cell viability was evaluated using the fluorescent dyes. The behavior of the cells was continuously monitored with time-lapse video microscopy. RESULTS: TQ significantly increased neuronal survival by promoting the survival and proliferation of SCs and fibroblasts, as well as the migration of SCs. Furthermore, TQ improved the ability to extend neurites of axotomized neurons. The regenerative effect of TQ was dose-dependent suggesting a target specificity. Our studies warrant further preclinical and clinical investigations of TQ as a potential regenerative agent to treat peripheral nerve injuries. CONCLUSION: TQ exhibits a regenerative potential for the treatment of damaged peripheral nerves.

Mimetics of suppressor of cytokine signaling 3: Novel potential therapeutics in triple breast cancer.

Suppressor of cytokine signaling (SOCS) family of proteins plays critical role in the regulation of immune responses controlling JAK/STAT mediated inflammatory cytokines. Among the members, SOCS1 and SOCS3 contain a kinase inhibitory region (KIR) and SOCS3 binds to JAK/STAT/gp130 complex by inhibiting the downstream signaling and suppressing inflammatory cytokines. Loss or reduced levels of SOCS3 have been linked to cancer-associated inflammation and suppressive immunity leading to enhanced tumor growth and metastasis. In line with these reports, we previously demonstrated that proteolytic degradation of SOCS3 in triple negative breast cancer (TNBC) subtype drives the expression of inflammatory cytokines. Therefore, we postulated that SOCS3 mimetics might suppress the inflammatory cytokine production in TNBC subtype and inhibit tumor growth and metastasis. Here we designed and characterized five linear peptides derived from the N-terminal region of SOCS3 encompassing regions that interface with the JAK2/gp130 complex using the Circular Dichroism and Surface Plasmon Resonance spectroscopies. The KIRESS peptide resulted the sequence containing the most part of the hot-spots required for binding to JAK2 and was further investigated in vivo in mouse xenografts of MDA-MB-231-luci tumors as models of human TNBC subtype. Expectedly, this peptide showed a significant inhibition of primary tumor growth and pulmonary metastasis. Our studies suggest that SOCS3 peptidomimetics may possess a therapeutic potential in aggressive cancers, such as TNBC subtype, with activated inflammatory cytokines.