Nimbolide Inhibits SOD2 to Control Pancreatic Ductal Adenocarcinoma Growth and Metastasis.

Reactive oxygen species are frequently associated with various cancers including pancreatic ductal adenocarcinomas (PDACs). Superoxide dismutase 2 (SOD2) is an enzyme that plays an important role in reactive oxygen species (ROS) signaling. Investigating the molecular function and biological functions of SOD2 can help us develop new therapeutic options and uncover new biomarkers for PDAC diagnosis and prognosis. Here, we show that nimbolide (NB), a triterpene limonoid, effectively blocks the growth and metastasis of PDACs by suppressing the expression and activity of SOD2. To identify the role of SOD2 in NB-induced anticancer activity, we used RNA interference to silence and plasmid transfection to overexpress it. Silencing SOD2 significantly reduced the growth and metastatic characteristics like epithelial-to-mesenchymal transition, invasion, migration, and colony-forming capabilities of PDACs, and NB treatment further reduced these characteristics. Conversely, the overexpression of SOD2 enhanced these metastatic characteristics. ROS signaling has a strong feedback mechanism with the PI3K/Akt signaling pathway, which could be mediated through SOD2. Finally, NB treatment to SOD2-overexpressing PDAC xenografts resulted in significant inhibition of tumor growth and metastasis. Overall, this work suggests that NB, a natural and safe phytochemical that silences SOD2 to induce high levels of ROS generation, results in increased apoptosis and reduced growth and progression of PDACs. The role of SOD2 in regulating NB-induced ROS generation presents itself as a therapeutic option for PDACs.

Metabolomics of Breast Cancer: A Review.

Breast cancer is the most commonly diagnosed cancer in women worldwide. Major advances have been made towards breast cancer prevention and treatment. Unfortunately, the incidence of breast cancer is still increasing globally. Metabolomics is the field of science which studies all the metabolites in a cell, tissue, system, or organism. Metabolomics can provide information on dynamic changes occurring during cancer development and progression. The metabolites identified using cutting-edge metabolomics techniques will result in the identification of biomarkers for the early detection, diagnosis, and treatment of cancers. This review briefly introduces the metabolic changes in cancer with particular focus on breast cancer.

Phytochemicals as an Alternative or Integrative Option, in Conjunction with Conventional Treatments for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most abundant form of liver cancer. It accounts for 75-85% of liver cancer cases and, though it ranks globally as the sixth most common cancer, it ranks second in cancer-related mortality. Deaths from HCC are usually due to metastatic spread of the cancer. Unfortunately, there are many challenges and limitations with the latest HCC therapies and medications, making it difficult for patients to receive life-prolonging care. As there is clearly a high demand for alternative therapy options for HCC, it is prudent to turn to plants for the solution, as their phytochemicals have long been used and revered for their many medicinal purposes. This review explores the promising phytochemical compounds identified from pre-clinical and clinical trials being used either independently or in conjunction with already existing cancer therapy treatments. The phytochemicals discussed in this review were classified into several categories: lipids, polyphenols, alkaloids, polysaccharides, whole extracts, and phytochemical combinations. Almost 80% of the compounds failed to progress into clinical studies due to lack of information regarding the toxicity to normal cells and bioavailability. Although large obstacles remain, phytochemicals can be used either as an alternative or integrative therapy in conjunction with existing HCC chemotherapies. In conclusion, phytochemicals have great potential as treatment options for hepatocellular carcinoma.

miRNome and Functional Network Analysis of PGRMC1 Regulated miRNA Target Genes Identify Pathways and Biological Functions Associated With Triple Negative Breast Cancer.

BACKGROUND: Increased expression of the progesterone receptor membrane component 1, a heme and progesterone binding protein, is frequently found in triple negative breast cancer tissue. The basis for the expression of PGRMC1 and its regulation on cellular signaling mechanisms remain largely unknown. Therefore, we aim to study microRNAs that target selective genes and mechanisms that are regulated by PGRMC1 in TNBCs. METHODS: To identify altered miRNAs, whole human miRNome profiling was performed following AG-205 treatment and PGRMC1 silencing. Network analysis identified miRNA target genes while KEGG, REACTOME and Gene ontology were used to explore altered signaling pathways, biological processes, and molecular functions. RESULTS: KEGG term pathway analysis revealed that upregulated miRNAs target specific genes that are involved in signaling pathways that play a major role in carcinogenesis. While multiple downregulated miRNAs are known oncogenes and have been previously demonstrated to be overexpressed in a variety of cancers. Overlapping miRNA target genes associated with KEGG term pathways were identified and overexpression/amplification of these genes was observed in invasive breast carcinoma tissue from TCGA. Further, the top two genes (CCND1 and YWHAZ) which are highly genetically altered are also associated with poorer overall survival. CONCLUSIONS: Thus, our data demonstrates that therapeutic targeting of PGRMC1 in aggressive breast cancers leads to the activation of miRNAs that target overexpressed genes and deactivation of miRNAs that have oncogenic potential.

Pregnancy Inhibits Mammary Carcinogenesis by Persistently Altering the Hypothalamic-Pituitary Axis.

Early full-term pregnancy is known to reduce the lifetime risk of breast cancer. Although the phenomenon of parity-induced protection is well-established, the physiological mechanisms involved in this protection are not clear. Earlier reports have shown that pregnancy results in alterations of hormone levels. How pregnancy affects hypothalamic hormones and how the change, if any, influences breast cancer is not well understood. Seven-week-old female Lewis rats were given N-methyl-N-nitrosourea. Two weeks post carcinogen exposure, a set of females were housed with males to generate the parous rats and another set of rats served as the nulliparous controls. Mammary tumorigenesis was assessed for 9 months. Hypothalamic and pituitary levels of hormones were measured at various timepoints. Further, animals were also challenged with growth hormone and prolactin secretagogues to test the effect of pregnancy on the hypothalamic-pituitary hormonal axis. Persistent alterations in the level of growth hormone-releasing hormone, thyrotropin releasing hormone, dopamine, and somatostatin in the hypothalamus of parous animals was observed. Further, we also observed that pregnancy had a significant effect on the pituitary gland and its response to growth hormone and prolactin secretagogues. Our studies using the rodent model system demonstrate that pregnancy could be reducing the risk of breast cancer by persistently altering the hypothalamic-pituitary axis, which could have implications for breast cancers in humans as well.

Crosstalk between progesterone receptor membrane component 1 and estrogen receptor α promotes breast cancer cell proliferation.

Progesterone (P4) and estradiol (E2) have been shown to stimulate and regulate breast cancer proliferation via classical nuclear receptor signaling through progesterone receptor (PR) and estrogen receptor α (ERα), respectively. However, the basis of communication between PR/ERα and membrane receptors remains largely unknown. Here, we aim to identify classical and nonclassical endocrine signaling mechanisms that can alter cell proliferation through a possible crosstalk between PR, ERα, and progesterone receptor membrane component 1 (PGRMC1), a membrane receptor frequently observed in breast cancer cells. While P4 and E2 treatment increased cell proliferation of ER+/PR+/PGRMC1 overexpressing breast cancer cells, silencing ERα and PR or treatment with selective estrogen receptor modulator (SERM) tamoxifen, or (PR-antagonist) RU-486 decreased cell proliferation. All four treatments rapidly altered PGRMC1 mRNA levels and protein expression. Furthermore, P4 and E2 treatments rapidly activated EGFR a known interacting partner of PGRMC1 and its downstream signaling. Interestingly, downregulation of ERα by tamoxifen and ERα silencing decreased the expression levels of PGRMC1 with no repercussions to PR expression. Strikingly PGRMC1 silencing decreased ERα expression irrespective of PR. METABRIC and TCGA datasets further demonstrated that PGRMC1 expression was comparable to that of ERα in Luminal A and B breast cancers. Targeting of PR, ERα, and PGRMC1 confirmed that a crosstalk between classical and nonclassical signaling mechanisms exists in ER+ breast cancer cells that could enhance the growth of ER+/PR+/PGRMC1 overexpressing tumors.

Involvement of actin cytoskeletal modifications in the inhibition of triple-negative breast cancer growth and metastasis by nimbolide.

Triple-negative breast cancers (TNBCs) are aggressive cancers, which currently do not have effective treatment options. Migration and establishment of metastatic colonies require dynamic cytoskeletal modifications characterized by polymerization and depolymerization of actin. Studies have demonstrated a direct molecular link between the integrin-focal adhesion kinase (FAK) pathway and cytoskeletal modifications. Nimbolide, a major bioactive compound present in neem leaves, shows promising anti-cancer effect on various cancers. In this study, we have demonstrated the growth and metastasis inhibitory potential of nimbolide on TNBC cells. Nimbolide inhibited cell proliferation, migratory, and invasive abilities of TNBC cells and also changed the shape of MDA-MB-231 cells, which is correlated with cytoskeletal changes including actin depolymerization. Furthermore, analysis revealed that integrins αV and ß3, ILK, FAK, and PAK levels were downregulated by nimbolide. Even in cells where Rac1/Cdc42 was constitutively activated, nimbolide inhibited the formation of filopodial structures. Immunofluorescence analysis of phosphorylated p21 activated kinase (pPAK) showed reduced expression in nimbolide-treated cells. Nimbolide significantly reduced the metastatic colony formation in lung, liver, and brain of athymic nude mice. In conclusion, our data demonstrate that nimbolide inhibits TNBC by altering the integrin and FAK signaling pathway.

Hepatocyte nuclear factor 1 alpha influences pancreatic cancer growth and metastasis.

Hepatocyte nuclear factor 1 homeobox alpha (HNF1α) is a transcription factor involved in endodermal organogenesis and pancreatic precursor cell differentiation and development. Earlier studies have reported a role for HNF1α in pancreatic ductal adenocarcinoma (PDAC) but it is controversial. The mechanism by which it impacts PDAC is yet to be explored in depth. In this study, using the online databases we observed that HNF1α is upregulated in PDAC, which was also confirmed by our immunohistochemical analysis of PDAC tissue microarray. Silencing HNF1α reduced the proliferative, migratory, invasive and colony forming capabilities of pancreatic cancer cells. Key markers involved in these processes (pPI3K, pAKT, pERK, Bcl2, Zeb, Snail, Slug) were significantly changed in response to alterations in HNF1α expression. On the other hand, overexpression of HNF1α did not induce any significant change in the aggressiveness of pancreatic cancer cells. Our results demonstrate that reduced expression of HNF1α leads to inhibition of pancreatic cancer growth and progression, which indicates that it could be a potential oncogene and target for PDAC.

Classical and Non-Classical Progesterone Signaling in Breast Cancers.

Much emphasis is placed on estrogen (E2) and estrogen receptor (ER) signaling as most research is focused on understanding E2 and ER's ability to enhance proliferative signals in breast cancers. Progesterone (P4) is important for normal mammary gland development, function and menstrual control. However, P4 and its receptors (PRs) in breast cancer etiology continue to be understudied and its role in breast cancer remains controversial. The Women's Health Initiative (WHI) clinical trial clearly demonstrated the importance of progestogens in breast cancer development. P4 has historically been associated with classical-signaling through nuclear receptors, however non-classical P4 signaling via membrane receptors has been described. Progestogens have the ability to bind to nuclear and membrane receptors and studies have demonstrated that both can promote breast cancer cell proliferation and breast tumor growth. In this review, we attempt to understand the classical and non-classical signaling role of P4 in breast cancers because both nuclear and membrane receptors could become viable therapeutic options for breast cancer patients.

Progesterone receptor membrane component 1 promotes the growth of breast cancers by altering the phosphoproteome and augmenting EGFR/PI3K/AKT signalling.

BACKGROUND: Increased expression of the progesterone receptor membrane component 1 (PGRMC1) has been linked to multiple cancers, including breast cancer. Despite being a regulatory receptor and a potential therapeutic target, the oncogenic potential of PGRMC1 has not been studied. METHODS: The impact of PGRMC1 on breast cancer growth and progression was studied following chemical inhibition and alteration of PGRMC1 expression, and evaluated by using online-based gene expression datasets of human breast cancer tissue. MTS, flow cytometry, qPCR, Western blotting, confocal microscopy and phosphoproteome analysis were performed. RESULTS: We observed higher PGRMC1 levels in both ER-positive ZR-75-1 and TNBC MDA-MB-468 cells. Both chemical inhibition and silencing decreased cell proliferation, induced cell-cycle arrest, promoted apoptosis and reduced the migratory and invasive capabilities of ZR-75-1 and MDA-MB-468 cells. Further, phosphoproteome analysis demonstrated an overall decrease in activation of proteins involved in PI3K/AKT/mTOR and EGFR signalling pathways. In contrast, overexpression of PGRMC1 in non-malignant MCF10A cells resulted in increased cell proliferation, and enhanced activity of PI3K/AKT/mTOR and EGFR signalling pathways. CONCLUSIONS: Our data demonstrate that PGRMC1 plays a prominent role in regulating the growth of cancer cells by altering the PI3K/AKT/mTOR and EGFR signalling mechanisms in both ER-positive and TNBC cells.

Hypoxanthine Phosphoribosyl Transferase 1 Is Upregulated, Predicts Clinical Outcome and Controls Gene Expression in Breast Cancer.

Hypoxanthine phosphoribosyl transferase 1 (HPRT1) is traditionally believed to be a housekeeping gene; however, recent reports suggest that it is upregulated in several cancers and is associated with clinical outcomes. HPRT1 is located on chromosome X and encodes the HPRT enzyme, which functions in recycling nucleotides to supply for DNA and RNA synthesis in actively dividing cells. Here, we used transcriptomic analyses to interrogate its expression across all known cancer types and elucidated its role in regulating gene expression in breast cancer. We observed elevated HPRT1 RNA levels in malignant tissues when compared to normal controls, indicating its potential as a diagnostic and prognostic marker. Further, in breast cancer, the subtype-specific analysis showed that its expression was highest in basal and triple-negative breast cancer, and HPRT1 knockdown in breast cancer cells suggested that HPRT1 positively regulates genes related to cancer pathways. Collectively, our results essentially highlight the importance of and change the way in which HPRT1's function is studied in biology, warranting careful examination of its role in cancer.

Long noncoding RNAs in cancer: From discovery to therapeutic targets.

Long noncoding RNAs (lncRNAs) have recently gained considerable attention as key players in biological regulation; however, the mechanisms by which lncRNAs govern various disease processes remain mysterious and are just beginning to be understood. The ease of next-generation sequencing technologies has led to an explosion of genomic information, especially for the lncRNA class of noncoding RNAs. LncRNAs exhibit the characteristics of mRNAs, such as polyadenylation, 5' methyl capping, RNA polymerase II-dependent transcription, and splicing. These transcripts comprise more than 200 nucleotides (nt) and are not translated into proteins. Directed interrogation of annotated lncRNAs from RNA-Seq datasets has revealed dramatic differences in their expression, largely driven by alterations in transcription, the cell cycle, and RNA metabolism. The fact that lncRNAs are expressed cell- and tissue-specifically makes them excellent biomarkers for ongoing biological events. Notably, lncRNAs are differentially expressed in several cancers and show a distinct association with clinical outcomes. Novel methods and strategies are being developed to study lncRNA function and will provide researchers with the tools and opportunities to develop lncRNA-based therapeutics for cancer.

Silencing growth hormone receptor inhibits estrogen receptor negative breast cancer through ATP-binding cassette sub-family G member 2.

Growth hormone receptor (GHR) plays a vital role in breast cancer chemoresistance and metastasis but the mechanism is not fully understood. We determined if GHR could be a potential therapeutic target for estrogen receptor negative (ER-ve) breast cancer, which are highly chemoresistant and metastatic. GHR was stably knocked down in ER-ve breast cancer cells and its effect on cell proliferation, metastatic behavior, and chemosensitivity to docetaxel (DT) was assessed. Microarray analysis was performed to identify potential GHR downstream targets involved in chemoresistance. GHR and ATP-binding cassette sub-family G member 2 (ABCG2) overexpression and knockdown studies were performed to investigate the mechanism of GHR-induced chemoresistance. Patient-derived xenografts was used to study the effect of GHR and ABCG2. Immunohistochemical data was used to determine the correlation between GHR, pAKT, pmTOR, and ABCG2 expressions. GHR silencing drastically reduced the chemoresistant and metastatic behavior of ER-ve breast cancer cells and also inhibited AKT/mTOR pathway. In contrast, activation, or overexpression of GHR increased chemoresistance and metastasis by increasing the expression and promoter activity, of ABCG2. Inhibition of JAK2/STAT5 signaling repressed GHR-induced ABCG2 promoter activity and expression. Further, ABCG2 knockdown significantly increased the chemosensitivity. Finally, patient-derived xenograft studies revealed the role of GHR in chemoresistance. Overall, these findings demonstrate that targeting GHR could be a novel therapeutic approach to overcome chemoresistance and associated metastasis in aggressive ER-ve breast cancers.

FOXC1 plays a crucial role in the growth of pancreatic cancer.

IGF-1R signaling controls various vital cellular functions and this signaling is deregulated in many cancers, including pancreatic cancer. Several efforts have mainly focused on inhibiting the IGF-1R signaling cascade. The outcomes of these focused preclinical studies have been positive, whereas clinical trials of IGF-1R inhibitors in pancreatic cancer have failed, raising the questions about this therapeutic approach. This necessitates a better understanding of the role of IGF-1R signaling in pancreatic cancer. We investigated the impact of IGF-1R signaling on crucial transcription factors and identified the FOXC1 as one of the crucial regulator of IGF-1R signaling. We employed genetic approaches to overexpress and silence FOXC1 in pancreatic cancer cells. Our results demonstrate that IGF-1R and FOXC1 seem to positively regulate each other. Further, FOXC1 increased the metastatic abilities of pancreatic cancer cells by enhancing cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, and angiogenesis. The data from xenograft experiments further established the importance of FOXC1 in pancreatic tumorigenesis. In conclusion, FOXC1 is a potent oncogenic transcription factor, which promotes pancreatic cancer growth and metastasis. Thus, targeting FOXC1 could be a potential therapeutic strategy against pancreatic cancer.

Complementary and Alternative Medicine and Breast Cancer.

Complementary and alternative medicine (CAM) has been in use among cancer patients for a long time. There are several types of CAM that are practiced in various parts of the world. For example, traditional medicinal practices followed in India and China are frequently used by cancer patients. CAM is broadly classified into five different categories: (1) traditional medicines, (2) mind-body interventions, (3) biology-based practices, (4) manipulative body-based practices, and (5) energy medicine. In this review, we have compiled data from the available literature regarding CAM use in breast cancer patients. We have highlighted the current concepts and the need for more structured studies to facilitate the implementation of CAM as a mainstream option for cancer patients.

Pregnancy and Breast Cancer.

Breast cancer is the most commonly diagnosed type of cancer among women worldwide. The majority of breast cancers are sporadic and the etiology is not well understood. Several factors have been attributed to altering the risk of breast cancer. A full-term pregnancy is a crucial factor in altering the risk. Early full-term pregnancy has been shown to reduce the lifetime risk of breast cancer, while a later first full-term pregnancy increases breast cancer risk. Epidemiological and experimental data demonstrate that spontaneous or induced abortions do not significantly alter the risk of breast cancer. In this study, we briefly discuss the different types and stages of breast cancer, various risk factors, and potential mechanisms involved in early full-term pregnancy-induced protection against breast cancer. Understanding how early full-term pregnancy induces protection against breast cancer will help design innovative preventive and therapeutic strategies. This understanding can also help in the development of molecular biomarkers that can be of tremendous help in predicting the risk of breast cancer in the general population.

Glucose insult elicits hyperactivation of cancer stem cells through miR-424-cdc42-prdm14 signalling axis.

BACKGROUND: Meta-analysis shows that women with diabetes have a 20% increased risk of breast cancer and also an increased risk for distant metastasis and mortality. The molecular mechanisms for distant metastasis and mortality in breast cancer patients with diabetes are not very well understood. METHODS: We compared the effect of physiological (5 mM) and diabetic (10 mM) levels of glucose on malignant breast epithelial cell invasion and stemness capabilities. We performed microRNA array to determine the dysregulated microRNAs in hyperglycaemic conditions and performed functional and molecular analysis of the gene targets. RESULTS: Hyperglycaemia leads to hyperactivation of cancer stem cell pool and enhances invasive ability of breast cancer cells. MiR-424 seems to be a key regulator of cancer cell stemness and invasion. Knockdown of miR-424 in cancer cells under euglycaemic conditions leads to enhanced invasion and stem cell activity, whereas ectopic expression of miR-424 in cancer cells under hyperglycaemic conditions results in suppressed invasion and stem cell activity. Cdc42, a target of miR-424, influences cancer stem cell activity by positively regulating prdm14 through activation of pak1 (p-21-activated kinase 1) and stat5. CONCLUSIONS: Our findings establish miR-424→︀cdc42→︀prdm14 axis as a key molecular signalling cascade that might influence breast cancer progression in diabetic patients through hyperactivation of cancer stem cells.

Role of Growth Hormone in Breast Cancer.

Breast cancer is one of the most common cancers diagnosed in women. Approximately two-thirds of all breast cancers diagnosed are classified as hormone dependent, which indicates that hormones are the key factors that drive the growth of these breast cancers. Ovarian and pituitary hormones play a major role in the growth and development of normal mammary glands and breast cancer. In particular, the effect of the ovarian hormone estrogen has received much attention in regard to breast cancer. Pituitary hormones prolactin and growth hormone have also been associated with breast cancer. Although the role of these pituitary hormones in breast cancers has been studied, it has not been investigated extensively. In this review, we attempt to compile basic information from most of the currently available literature to understand and demonstrate the significance of growth hormone in breast cancer. Based on the available literature, it is clear that growth hormone plays a significant role in the development, progression, and metastasis of breast cancer by influencing tumor angiogenesis, stemness, and chemoresistance.

Gedunin inhibits pancreatic cancer by altering sonic hedgehog signaling pathway.

INTRODUCTION: The lack of efficient treatment options for pancreatic cancer highlights the critical need for the development of novel and effective chemotherapeutic agents. The medicinal properties found in plants have been used to treat many different illnesses including cancers. This study focuses on the anticancer effects of gedunin, a natural compound isolated from Azadirachta indica. METHODS: Anti-proliferative effect of gedunin on pancreatic cancer cells was assessed using MTS assay. We used matrigel invasion assay, scratch assay, and soft agar colony formation assay to measure the anti-metastatic potential of gedunin. Immunoblotting was performed to analyze the effect of gedunin on the expression of key proteins involved in pancreatic cancer growth and metastasis. Gedunin induced apoptosis was measured using flow cytometric analysis. To further validate, xenograft studies with HPAC cells were performed. RESULTS: Gedunin treatment is highly effective in inducing death of pancreatic cancer cells via intrinsic and extrinsic mediated apoptosis. Our data further indicates that gedunin inhibited metastasis of pancreatic cancer cells by decreasing their EMT, invasive, migratory and colony formation capabilities. Gedunin treatment also inhibited sonic hedgehog signaling pathways. Further, experiments with recombinant sonic hedgehog protein and Gli inhibitor (Gant-61) demonstrated that gedunin induces its anti-metastatic effect through inhibition of sonic hedgehog signaling. The anti-cancer effect of gedunin was further validated using xenograft mouse model. CONCLUSION: Overall, our data suggests that gedunin could serve as a potent anticancer agent against pancreatic cancers.

The serum protein profile of early parity which induces protection against breast cancer.

Early parity reduces the risk of breast cancer in women while nulliparity and late parity increase the risk of breast cancer. In order to translate this protection to women where early pregnancy is not feasible, much work has focused on understanding how parity confers protection against breast cancer, the molecular mechanisms by which this occurs is still not well understood. Healthy parous and nulliparous women were recruited for this study. We assessed serum protein profiles of early parous, late parous, and nulliparous women using the Phospho Explorer antibody array. Significantly altered proteins identified were validated by Western blot analysis. In silico analysis was performed with the data obtained. Our findings indicate increased phosphorylation levels of CDK1, AKT1 and Epo-R increased cell cycle and cell proliferation in late/nulliparous women. Increased levels of LIMK1, paxillin, caveolin-1, and tyrosine hydroxylase in late/nulliparous women demonstrate enhanced cell stress while decreased activity of p-p53 and pRAD51 in late/nulliparous women indicates decreased apoptosis and increased genomic instability. Further, increased levels of pFAK, pCD3zeta, pSTAT5B, MAP3K8 in early parous women favor enhanced innate/adaptive immunity. Overall, we have identified a unique protein signature that is responsible for the decreased risk of breast cancer and these proteins can also serve as biomarkers to predict the risk of breast cancer.