Tumor microenvironment and epithelial mesenchymal transition as targets to overcome tumor multidrug resistance.

It is well established that multifactorial drug resistance hinders successful cancer treatment. Tumor cell interactions with the tumor microenvironment (TME) are crucial in epithelial-mesenchymal transition (EMT) and multidrug resistance (MDR). TME-induced factors secreted by cancer cells and cancer-associated fibroblasts (CAFs) create an inflammatory microenvironment by recruiting immune cells. CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) and inflammatory tumor associated macrophages (TAMs) are main immune cell types which further enhance chronic inflammation. Chronic inflammation nurtures tumor-initiating/cancer stem-like cells (CSCs), induces both EMT and MDR leading to tumor relapses. Pro-thrombotic microenvironment created by inflammatory cytokines and chemokines from TAMs, MDSCs and CAFs is also involved in EMT and MDR. MDSCs are the most common mediators of immunosuppression and are also involved in resistance to targeted therapies, e.g. BRAF inhibitors and oncolytic viruses-based therapies. Expansion of both cancer and stroma cells causes hypoxia by hypoxia-inducible transcription factors (e.g. HIF-1α) resulting in drug resistance. TME factors induce the expression of transcriptional EMT factors, MDR and metabolic adaptation of cancer cells. Promoters of several ATP-binding cassette (ABC) transporter genes contain binding sites for canonical EMT transcription factors, e.g. ZEB, TWIST and SNAIL. Changes in glycolysis, oxidative phosphorylation and autophagy during EMT also promote MDR. Conclusively, EMT signaling simultaneously increases MDR. Owing to the multifactorial nature of MDR, targeting one mechanism seems to be non-sufficient to overcome resistance. Targeting inflammatory processes by immune modulatory compounds such as mTOR inhibitors, demethylating agents, low-dosed histone deacetylase inhibitors may decrease MDR. Targeting EMT and metabolic adaptation by small molecular inhibitors might also reverse MDR. In this review, we summarize evidence for TME components as causative factors of EMT and anticancer drug resistance.

Repurposing old drugs to fight multidrug resistant cancers.

Overcoming multidrug resistance represents a major challenge for cancer treatment. In the search for new chemotherapeutics to treat malignant diseases, drug repurposing gained a tremendous interest during the past years. Repositioning candidates have often emerged through several stages of clinical drug development, and may even be marketed, thus attracting the attention and interest of pharmaceutical companies as well as regulatory agencies. Typically, drug repositioning has been serendipitous, using undesired side effects of small molecule drugs to exploit new disease indications. As bioinformatics gain increasing popularity as an integral component of drug discovery, more rational approaches are needed. Herein, we show some practical examples of in silico approaches such as pharmacophore modelling, as well as pharmacophore- and docking-based virtual screening for a fast and cost-effective repurposing of small molecule drugs against multidrug resistant cancers. We provide a timely and comprehensive overview of compounds with considerable potential to be repositioned for cancer therapeutics. These drugs are from diverse chemotherapeutic classes. We emphasize the scope and limitations of anthelmintics, antibiotics, antifungals, antivirals, antimalarials, antihypertensives, psychopharmaceuticals and antidiabetics that have shown extensive immunomodulatory, antiproliferative, pro-apoptotic, and antimetastatic potential. These drugs, either used alone or in combination with existing anticancer chemotherapeutics, represent strong candidates to prevent or overcome drug resistance. We particularly focus on outcomes and future perspectives of drug repositioning for the treatment of multidrug resistant tumors and discuss current possibilities and limitations of preclinical and clinical investigations.

Matrikine and matricellular regulators of EGF receptor signaling on cancer cell migration and invasion.

Cancer invasion is a complex process requiring, among other events, extensive remodeling of the extracellular matrix including deposition of pro-migratory and pro-proliferative moieties. In recent years, it has been described that while invading through matrices cancer cells can change shape and adapt their migration strategies depending on the microenvironmental context. Although intracellular signaling pathways governing the mesenchymal to amoeboid migration shift and vice versa have been mostly elucidated, the extracellular signals promoting these shifts are largely unknown. In this review, we summarize findings that point to matrikines that bind specifically to the EGF receptor as matricellular molecules that enable cancer cell migrational plasticity and promote invasion.

Pioglitazone decreases portosystemic shunting by modulating inflammation and angiogenesis in cirrhotic and non-cirrhotic portal hypertensive rats.

BACKGROUND & AIMS: Development of the portal-hypertensive syndrome is mediated by splanchnic inflammation and neoangiogenesis. Since peroxisome proliferator-activated receptor gamma (PPARγ) agonists like pioglitazone (PIO) regulate inflammatory response and inhibit angiogenesis in endothelial cells, we evaluated PIO as treatment for experimental portal hypertension. METHODS: PIO (10 mg/kg) or vehicle (VEH) was administered from day 21-28 after bile duct ligation (BDL), from day 0-7 after partial portal vein ligation (PPVL) or sham-operation (SO), respectively. After treatment, systemic hemodynamics, splanchnic blood flow (SMABF), portal pressure (PP), and portosystemic shunting (PSS) were assessed. Splanchnic and hepatic tissues were analyzed for angiogenic and inflammatory markers. RESULTS: BDL and PPVL showed significantly increased PP, SMABF, and PSS compared to SO-VEH rats. While PIO treatment did not decrease PP or SMABF, PSS was significantly reduced both in cirrhotic (BDL-VEH: 71% to BDL-PIO: 41%; p<0.001) and non-cirrhotic (PPVL-VEH: 62% to PPVL-PIO: 40%; p=0.041) rats. PIO (10 µM, in vitro) inhibited endothelial cell migration and significantly increased PPARγ activity in vivo. In BDL rats, PIO decreased hepatic mRNA levels of PPARγ (p=0.01) and PlGF (p=0.071), and splanchnic mRNA expression of PPARγ (p=0.017), PDGFß (p=0.053) and TNFα (p=0.075). Accordingly, splanchnic protein expression of PPARγ (p=0.032), VEGFR2 (p=0.035), CD31 (p=0.060) and PDGFß (p=0.066) were lower in BDL-PIO vs. BDL-VEH animals. In PPVL rats, PIO treatment decreased splanchnic gene expression of Ang2 (-12.4 fold), eNOS (-9.3 fold), PDGF (-7.0 fold), PlGF (-11.9 fold), TGFb (-8.3 fold), VEGF-A (-11.3 fold), VEGFR1 (-5.9 fold), IL1b (-14.4 fold), and IL6 (-9.6 fold). CONCLUSIONS: Pioglitazone treatment decreases portosystemic shunting via modulation of splanchnic inflammation and neoangiogenesis. Pioglitazone should be assessed for potential beneficial effects in patients with portosystemic collaterals due to portal hypertension.

Analysis of the nischarin expression across human tumor types reveals its context-dependent role and a potential as a target for drug repurposing in oncology.

Nischarin was reported to be a tumor suppressor that plays a critical role in breast cancer initiation and progression, and a positive prognostic marker in breast, ovarian and lung cancers. Our group has found that nischarin had positive prognostic value in female melanoma patients, but negative in males. This opened up a question whether nischarin has tumor type-specific and sex-dependent roles in cancer progression. In this study, we systematically examined in the public databases the prognostic value of nischarin in solid tumors, regulation of its expression and associated signaling pathways. We also tested the effects of a nischarin agonist rilmenidine on cancer cell viability in vitro. Nischarin expression was decreased in tumors compared to the respective healthy tissues, most commonly due to the deletions of the nischarin gene and promoter methylation. Unlike in healthy tissues where it was located in the cytoplasm and at the membrane, in tumor tissues nischarin could also be observed in the nuclei, implying that nuclear translocation may also account for its cancer-specific role. Surprisingly, in several cancer types high nischarin expression was a negative prognostic marker. Gene set enrichment analysis showed that in tumors in which high nischarin expression was a negative prognostic marker, signaling pathways that regulate stemness were enriched. In concordance with the findings that nischarin expression was negatively associated with pathways that control cancer growth and progression, nischarin agonist rilmenidine decreased the viability of cancer cells in vitro. Taken together, our study lays a ground for functional studies of nischarin in a context-dependent manner and, given that nischarin has several clinically approved agonists, provides rationale for their repurposing, at least in tumors in which nischarin is predicted to be a positive prognostic marker.

Nischarin expression may have differing roles in male and female melanoma patients.

Due to the development of resistance to previously effective therapies, there is a constant need for novel treatment modalities for metastatic melanoma. Nischarin (NISCH) is a druggable scaffolding protein reported as a tumor suppressor and a positive prognostic marker in breast and ovarian cancers through regulation of cancer cell survival, motility and invasion. The aim of this study was to examine the expression and potential role of nischarin in melanoma. We found that nischarin expression was decreased in melanoma tissues compared to the uninvolved skin, and this was attributed to the presence of microdeletions and hyper-methylation of the NISCH promoter in the tumor tissue. In addition to the previously reported cytoplasmic and membranous localization, we observed nischarin in the nuclei in melanoma patients' tissues. NISCH expression in primary melanoma had favorable prognostic value for female patients, but, unexpectedly, high NISCH expression predicted worse prognosis for males. Gene set enrichment analysis suggested significant sex-related disparities in predicted association of NISCH with several signaling pathways, as well as with different tumor immune infiltrate composition in male and female patients. Taken together, our results imply that nischarin may have a role in melanoma progression, but that fine-tuning of the pathways it regulates is sex-dependent. KEY MESSAGES: Nischarin is a tumor suppressor whose role has not been investigated in melanoma. Nischarin expression was downregulated in melanoma tissue compared to the normal skin. Nischarin had the opposite prognostic value in male and female melanoma patients. Nischarin association with signaling pathways differed in females and males. Our findings challenge the current view of nischarin as a universal tumor suppressor.

Prognostic Value of Combined Hematological/Biochemical Indexes and Tumor Clinicopathologic Features in Colorectal Cancer Patients-A Pilot Single Center Study.

Colorectal cancer (CRC) is a significant public health problem. There is increasing evidence that the host's immune response and nutritional status play a role in the development and progression of cancer. The aim of our study was to examine the prognostic value of clinical markers/indexes of inflammation, nutritional and pathohistological status in relation to overall survival and disease free-survival in CRC. The total number of CRC patients included in the study was 111 and they underwent laboratory analyses within a week before surgery. Detailed pathohistological analysis and laboratory parameters were part of the standard hospital pre-operative procedure. Medical data were collected from archived hospital data. Data on the exact date of death were obtained by inspecting the death registers for the territory of the Republic of Serbia. All parameters were analyzed in relation to the overall survival and survival period without disease relapse. The follow-up median was 42 (24-48) months. The patients with the III, IV and V degrees of the Clavien-Dindo classification had 2.609 (HR: 2.609; 95% CI: 1.437-4.737; p = 0.002) times higher risk of death. The modified Glasgow prognostic score (mGPS) 2 and higher lymph node ratio carried a 2.188 (HR: 2.188; 95% CI: 1.413-3.387; p < 0.001) and 6.862 (HR: 6.862; 95% CI: 1.635-28.808; p = 0.009) times higher risk of death in the postoperative period, respectively; the risk was 3.089 times higher (HR: 3.089; 95% CI: 1.447-6.593; p = 0.004) in patients with verified tumor deposits. The patients with tumor deposits had 1.888 (HR: 1.888; 95% CI: 1024-3481; p = 0.042) and 3.049 (HR: 3.049; 95% CI: 1.206-7.706; p = 0.018) times higher risk of disease recurrence, respectively. The emphasized peritumoral lymphocyte response reduced the risk of recurrence by 61% (HR: 0.391; 95% CI: 0.196-0.780; p = 0.005). Standard perioperative laboratory and pathohistological parameters, which do not present any additional cost for the health system, may provide information on the CRC patient outcome and lay the groundwork for a larger prospective examination.

A Fluorescence-Based Assay for Measuring Glucose Uptake in Living Melanoma Cells.

Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.

Proteomic analysis of laser microdissected melanoma cells from skin organ cultures.

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma and understanding how the local microenvironment at the melanoma site influences this progression are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs) and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests were analyzed by nanoflow liquid chromatography-tandem mass spectrometry. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, noninvolved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment.

Synthesis and evaluation of anticancer activity of new 9-acridinyl amino acid derivatives.

A series of eleven 9-acridinyl amino acid derivatives were synthesized using a two-step procedure. Cytotoxicity was tested on the K562 and A549 cancer cell lines and normal diploid cell line MRC5 using the MTT assay. Compounds 6, 7, 8 and 9 were the most active, with IC50 values comparable to or lower than that of chemotherapeutic agent amsacrine. 8 and 9 were especially effective in the A549 cell line (IC50 ≈ 6 µM), which is of special interest since amsacrine is not sufficiently active in lung cancer patients. Cell cycle analysis revealed that 7 and 9 caused G2/M block, amsacrine caused arrest in the S phase, while 6 and 8 induced apoptotic cell death independently of the cell cycle regulation. In comparison to amsacrine, 6, 7, 8, and 9 showed similar inhibitory potential towards topoisomerase II, whereas only 7 showed DNA intercalation properties. In contrast to amsacrine, 6, 7, 8 and 9 showed a lack of toxicity towards unstimulated normal human leucocytes.

Telmisartan induces melanoma cell apoptosis and synergizes with vemurafenib in vitro by altering cell bioenergetics.

OBJECTIVE: Despite recent advancements in targeted therapy and immunotherapies, prognosis for metastatic melanoma patients remains extremely poor. Development of resistance to previously effective treatments presents a serious challenge and new approaches for melanoma treatment are urgently needed. The objective of this study was to examine the effects of telmisartan, an AGTR1 inhibitor and a partial agonist of PPARγ, on melanoma cells as a potential agent for repurposing in melanoma treatment. METHODS: Expression of AGTR1 and PPARγ mRNA in melanoma patient tumor samples was examined in publicly available datasets and confirmed in melanoma cell lines by qRT-PCR. A panel of melanoma cell lines was tested in viability, apoptosis and metabolic assays in presence of telmisartan by flow cytometry and immunocytochemistry. A cytotoxic effect of combinations of telmisartan and targeted therapy vemurafenib was examined using the Chou-Talalay combination index method. RESULTS: Both AGTR1 and PPARγ mRNA were expressed in melanoma patient tumor samples and decreased compared to the expression in the healthy skin. In vitro, we found that telmisartan decreased melanoma cell viability by inducing cell apoptosis. Increased glucose uptake, but not utilization, in the presence of telmisartan caused the fission of mitochondria and release of reactive oxygen species. Telmisartan altered the cell bioenergetics, thereby synergizing with vemurafenib in vitro, and even sensitized vemurafenib-resistant cells to the treatment. CONCLUSIONS: Given that the effective doses of telmisartan examined in our study can be administered to patients and that telmisartan is a widely used and safe antihypertensive drug, our findings provide the scientific rationale for testing its efficacy in treatment of melanoma progression.

Vascular beds maintain pancreatic tumour explants for ex vivo drug screening.

Our understanding of cancer progression or response to therapies would benefit from benchtop, tissue-level assays that preserve the biology and anatomy of human tumours ex vivo. We present a methodology for maintaining patient tumour samples ex vivo for the purpose of drug testing in a clinical setting. The harvested tumour biopsy, excised from mice or patients, is integrated into a support tissue that includes stroma and vasculature. This support tissue preserves tumour histoarchitecture and relevant expression profiles, and tumour tissues cultured using this system display different sensitivities to chemotherapeutics compared with tumour explants with no supporting tissue. The methodology is more rapid than patient-derived xenograft models, easy to implement, and amenable to high-throughput assays, making it an attractive tool for in vitro drug screening or for the guidance of patient-specific chemotherapies.

The crosstalk between breast carcinoma-associated fibroblasts and cancer cells promotes RhoA-dependent invasion via IGF-1 and PAI-1.

Carcinoma-associated fibroblasts (CAFs) can remodel the extracellular matrix to promote cancer cell invasion, but the paracrine signaling between CAFs and cancer cells that regulates tumor cell migration remains to be identified. To determine how the interaction between CAFs and cancer cells modulates the invasiveness of cancer cells, we developed a 3-dimensional co-culture model composed of breast cancer (BC) MDA-MB-231 cell spheroids embedded in a collagen gel with and without CAFs. We found that the crosstalk between CAFs and cancer cells promotes invasion by stimulating the scattering of MDA-MB-231 cells, which was dependent on RhoA/ROCK/phospho MLC signaling in cancer cells but independent of RhoA in CAFs. The activation of RhoA/ROCK in cancer cells activates MLC and increases migration, while the genetic-down-regulation of RhoA and pharmacological inhibition of ROCK reduced cell scattering and invasion. Two distinct mechanisms induced the activation of the RhoA/ROCK pathway in MDA-MB-231 cells, the secretion of IGF-1 by CAFs and the upregulation of PAI-1 in cancer cells. In an orthotopic model of BC, IGF-1R inhibition decreased the incidence of lung metastasis, while Y27632-inhibition of ROCK enhanced the lung metastasis burden, which was associated with an increased recruitment of CAFs and expression of PAI-1. Thus the crosstalk between CAFs and BC cells increases the secretion of IGF-1 in CAFs and PAI-1 activity in cancer cells. Both IGF1 and PAI-1 activate RhoA/ROCK signaling in cancer cells, which increases cell scattering and invasion.

Obesity-Induced Inflammation and Desmoplasia Promote Pancreatic Cancer Progression and Resistance to Chemotherapy.

UNLABELLED: It remains unclear how obesity worsens treatment outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). In normal pancreas, obesity promotes inflammation and fibrosis. We found in mouse models of PDAC that obesity also promotes desmoplasia associated with accelerated tumor growth and impaired delivery/efficacy of chemotherapeutics through reduced perfusion. Genetic and pharmacologic inhibition of angiotensin-II type-1 receptor reverses obesity-augmented desmoplasia and tumor growth and improves response to chemotherapy. Augmented activation of pancreatic stellate cells (PSC) in obesity is induced by tumor-associated neutrophils (TAN) recruited by adipocyte-secreted IL1ß. PSCs further secrete IL1ß, and inactivation of PSCs reduces IL1ß expression and TAN recruitment. Furthermore, depletion of TANs, IL1ß inhibition, or inactivation of PSCs prevents obesity-accelerated tumor growth. In patients with pancreatic cancer, we confirmed that obesity is associated with increased desmoplasia and reduced response to chemotherapy. We conclude that cross-talk between adipocytes, TANs, and PSCs exacerbates desmoplasia and promotes tumor progression in obesity. SIGNIFICANCE: Considering the current obesity pandemic, unraveling the mechanisms underlying obesity-induced cancer progression is an urgent need. We found that the aggravation of desmoplasia is a key mechanism of obesity-promoted PDAC progression. Importantly, we discovered that clinically available antifibrotic/inflammatory agents can improve the treatment response of PDAC in obese hosts. Cancer Discov; 6(8); 852-69. ©2016 AACR.See related commentary by Bronte and Tortora, p. 821This article is highlighted in the In This Issue feature, p. 803.

Melanoma cell invasiveness is promoted at least in part by the epidermal growth factor-like repeats of tenascin-C.

Tenascin-C (TNC), overexpressed in invasive growths, has been implicated in progression of melanoma, but the source and function of this molecule are not well defined. We found TNC expression at the front of invading melanoma cells, and that adding TNC to matrices enhances individual melanoma cell migration. As TNC is a multidomain protein, we examined the role of the TNC EGF-like (EGFL) repeats as these activate motogenic signaling cascades. We overexpressed a TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). TNCEGFL-expressing melanoma cells had lower speed and persistence in 2D migration assays due to a shift in the adhesion-contractility balance, as expression of TNCEGFL delayed melanoma cell attachment and spreading. The less adhesive phenotype was due, in part, to increased Rho-associated kinase (ROCK) signaling concomitant with myosin light chain 2 and MYPT phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL-expressing melanoma cells and increased their migration in 2D. In contrast to the diminished migration in 2D, TNCEGFL-expressing melanoma cells had higher invasive potential in Matrigel invasion assays, with cells expressing TNCEGFL having amoeboid morphology. Our findings suggest that melanoma-derived TNCEGFL exert a role in melanoma invasion by modulating ROCK signaling and cell migration.

Targeting tumor cell motility as a strategy against invasion and metastasis.

Advances in diagnosis and treatment have rendered most solid tumors largely curable if they are diagnosed and treated before dissemination. However, once they spread beyond the initial primary location, these cancers are usually highly morbid, if not fatal. Thus, current efforts focus on both limiting initial dissemination and preventing secondary spread. There are two modes of tumor dissemination - invasion and metastasis - each leading to unique therapeutic challenges and likely to be driven by distinct mechanisms. However, these two forms of dissemination utilize some common strategies to accomplish movement from the primary tumor, establishment in an ectopic site, and survival therein. The adaptive behaviors of motile cancer cells provide an opening for therapeutic approaches if we understand the molecular, cellular, and tissue biology that underlie them. Herein, we review the signaling cascades and organ reactions that lead to dissemination, as these are non-genetic in nature, focusing on cell migration as the key to tumor progression. In this context, the cellular phenotype will also be discussed because the modes of migration are dictated by quantitative and physical aspects of the cell motility machinery.

Epithelial and mesenchymal phenotypic switchings modulate cell motility in metastasis.

The most ominous stage of cancer progression is metastasis, or the dissemination of carcinoma cells from the primary site into distant organs. Metastases are often resistant to current extirpative therapies and even the newest biological agents cure only a small subset of patients. Therefore a greater understanding of tumor biology that integrates properties intrinsic to carcinomas with tissue environmental modulators of behavior is needed. In no aspect of tumor progression is this more evident than the acquisition of cell motility that is critical for both escape from the primary tumor and colonization. In this overview, we discuss how this behavior is modified by carcinoma cell phenotypic plasticity that is evidenced by reversible switching between epithelial and mesenchymal phenotypes. The presence or absence of intercellular adhesions mediate these switches and dictate the receptivity towards signals from the extracellular milieu. These signals, which include soluble growth factors, cytokines, and extracellular matrix embedded with matrikines and matricryptines will be discussed in depth. Finally, we will describe a new mode of discerning the balance between epithelioid and mesenchymal movement.