Analysis of solid tumor mutation profiles in liquid biopsy.

Liquid biopsy is increasingly gaining traction as an alternative to invasive solid tumor biopsies for prognosis, treatment decisions, and disease monitoring. Matched tumor-plasma samples were collected from 180 patients across different cancers with >90% of the samples below Stage IIIB. Tumors were profiled using next-generation sequencing (NGS) or quantitative PCR (qPCR), and the mutation status was queried in the matched plasma using digital platforms such as droplet digital PCR (ddCPR) or NGS for concordance. Tumor-plasma concordance of 82% and 32% was observed in advanced (Stage IIB and above) and early (Stage I to Stage IIA) stage samples, respectively. Interestingly, the overall survival outcomes correlated to presurgical/at-biopsy ctDNA levels. Baseline ctDNA stratified patients into three categories: (a) high ctDNA correlated with poor survival outcome, (b) undetectable ctDNA with good outcome, and (c) low ctDNA whose outcome was ambiguous. ctDNA could be a powerful tool for therapy decisions and patient management in a large number of cancers across a variety of stages.

AMP-activated protein kinase promotes epithelial-mesenchymal transition in cancer cells through Twist1 upregulation.

The developmental programme of epithelial-mesenchymal transition (EMT), involving loss of epithelial and acquisition of mesenchymal properties, plays an important role in the invasion-metastasis cascade of cancer cells. In the present study, we show that activation of AMP-activated protein kinase (AMPK) using A769662 led to a concomitant induction of EMT in multiple cancer cell types, as observed by enhanced expression of mesenchymal markers, decrease in epithelial markers, and increase in migration and invasion. In contrast, inhibition or depletion of AMPK led to a reversal of EMT. Importantly, AMPK activity was found to be necessary for the induction of EMT by physiological cues such as hypoxia and TGFß treatment. Furthermore, AMPK activation increased the expression and nuclear localization of Twist1, an EMT transcription factor. Depletion of Twist1 impaired AMPK-induced EMT phenotypes, suggesting that AMPK might mediate its effects on EMT, at least in part, through Twist1 upregulation. Inhibition or depletion of AMPK also attenuated metastasis. Thus, our data underscore a central role for AMPK in the induction of EMT and in metastasis, suggesting that strategies targeting AMPK might provide novel approaches to curb cancer spread.

AMPK-Akt Double-Negative Feedback Loop in Breast Cancer Cells Regulates Their Adaptation to Matrix Deprivation.

Cell detachment from the extracellular matrix triggers anoikis. Disseminated tumor cells must adapt to survive matrix deprivation, while still retaining the ability to attach at secondary sites and reinitiate cell division. In this study, we elucidate mechanisms that enable reversible matrix attachment by breast cancer cells. Matrix deprival triggered AMPK activity and concomitantly inhibited AKT activity by upregulating the Akt phosphatase PHLPP2. The resultant pAMPKhigh/pAktlow state was critical for cell survival in suspension, as PHLPP2 silencing also increased anoikis while impairing autophagy and metastasis. In contrast, matrix reattachment led to Akt-mediated AMPK inactivation via PP2C-α-mediated restoration of the pAkthigh/pAMPKlow state. Clinical specimens of primary and metastatic breast cancer displayed an Akt-associated gene expression signature, whereas circulating breast tumor cells displayed an elevated AMPK-dependent gene expression signature. Our work establishes a double-negative feedback loop between Akt and AMPK to control the switch between matrix-attached and matrix-detached states needed to coordinate cell growth and survival during metastasis.Significance: These findings reveal a molecular switch that regulates cancer cell survival during metastatic dissemination, with the potential to identify targets to prevent metastasis in breast cancer. Cancer Res; 78(6); 1497-510. ©2018 AACR.

Corrigendum: A dual specificity kinase, DYRK1A, as a potential therapeutic target for head and neck squamous cell carcinoma.

This corrects the article DOI: 10.1038/srep36132.

StrandAdvantage test for early-line and advanced-stage treatment decisions in solid tumors.

Comprehensive genetic profiling of tumors using next-generation sequencing (NGS) is gaining acceptance for guiding treatment decisions in cancer care. We designed a cancer profiling test combining both deep sequencing and immunohistochemistry (IHC) of relevant cancer targets to aid therapy choices in both standard-of-care (SOC) and advanced-stage treatments for solid tumors. The SOC report is provided in a short turnaround time for four tumors, namely lung, breast, colon, and melanoma, followed by an investigational report. For other tumor types, an investigational report is provided. The NGS assay reports single-nucleotide variants (SNVs), copy number variations (CNVs), and translocations in 152 cancer-related genes. The tissue-specific IHC tests include routine and less common markers associated with drugs used in SOC settings. We describe the standardization, validation, and clinical utility of the StrandAdvantage test (SA test) using more than 250 solid tumor formalin-fixed paraffin-embedded (FFPE) samples and control cell line samples. The NGS test showed high reproducibility and accuracy of >99%. The test provided relevant clinical information for SOC treatment as well as more information related to investigational options and clinical trials for >95% of advanced-stage patients. In conclusion, the SA test comprising a robust and accurate NGS assay combined with clinically relevant IHC tests can detect somatic changes of clinical significance for strategic cancer management in all the stages.

Expression of tripartite motif-containing protein 28 in primary breast carcinoma predicts metastasis and is involved in the stemness, chemoresistance, and tumor growth.

The prediction of who develops metastasis has been the most difficult aspect in the management of breast cancer patients. The lymph node metastasis has been the most useful predictor of prognosis and patient management. However, a good proportion of patients with lymph node positivity remain disease free for 5 years or more, while about a third of those who were lymph node negative develop distant metastasis within the same period. This warrants a robust biomarker(s), preferably gene expression based. In order to elucidate gene-based biomarkers for prognosis of breast cancers, gene expression profiling of primary tumors and follow-up for over 5 years has been performed. The analysis revealed a network of genes centered around the tripartite motif-containing protein 28 as an important indicator of disease progression. Short hairpin RNA-mediated knockdown of tripartite motif-containing protein 28 in breast cancer cells revealed a decreased expression of epithelial-to-mesenchymal transition markers and increased expression of epithelial markers, decreased migration and invasion, and increased chemosensitivity to doxorubicin, 5-fluorouracil, and methotrexate. Furthermore, knockdown of tripartite motif-containing protein 28 resulted in the decrease of stemness as revealed by sphere formation assay as well as decreased expression of CD44 and Bmi1. Moreover, tripartite motif-containing protein 28 knockdown significantly reduced the tumor size and lung metastasis in orthotopic tumor xenograft assay in immunocompromised mice. The tumor size was further reduced when these mice were treated with doxorubicin. These data provide evidence for tripartite motif-containing protein 28 as a biomarker and a potential therapeutic target for breast cancer.

Different Phases of Breast Cancer Cells: Raman Study of Immortalized, Transformed, and Invasive Cells.

Breast cancer is the most prevalent cause of cancer-associated death in women the world over, but if detected early it can be treated successfully. Therefore, it is important to diagnose this disease at an early stage and to understand the biochemical changes associated with cellular transformation and cancer progression. Deregulated lipid metabolism has been shown to contribute to cell transformation as well as cancer progression. In this study, we monitored the biomolecular changes associated with the transformation of a normal cell into an invasive cell associated with breast cancer using Raman microspectroscopy. We have utilized primary normal breast cells, and immortalized, transformed, non-invasive, and invasive breast cancer cells. The Raman spectra were acquired from all these cell lines under physiological conditions. The higher wavenumber (2800-3000 cm-1) and lower wavenumber (700-1800 cm-1) range of the Raman spectrum were analyzed and we observed increased lipid levels for invasive cells. The Raman spectral data were analyzed by principal component-linear discriminant analysis (PC-LDA), which resulted in the formation of distinct clusters for different cell types with a high degree of sensitivity. The subsequent testing of the PC-LDA analysis via the leave-one-out cross validation approach (LOOCV) yielded relatively high identification sensitivity. Additionally, the Raman spectroscopic results were confirmed through fluorescence staining tests with BODIPY and Nile Red biochemical assays. Furthermore, Raman maps from the above mentioned cells under fixed conditions were also acquired to visualize the distribution of biomolecules throughout the cell. The present study shows the suitability of Raman spectroscopy as a non-invasive, label-free, microspectroscopic technique, having the potential of probing changes in the biomolecular composition of living cells as well as fixed cells.

A dual specificity kinase, DYRK1A, as a potential therapeutic target for head and neck squamous cell carcinoma.

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells.

Epidemiological data clearly establishes cigarette smoking as one of the major cause for lung cancer worldwide. Recently, targeted therapy has become one of the most preferred modes of treatment for cancer. Though certain targeted therapies such as anti-EGFR are in clinical practice, they have shown limited success in lung cancer patients who are smokers. This demands discovery of alternative drug targets through systematic investigation of cigarette smoke-induced signaling mechanisms. To study the signaling events activated in response to cigarette smoke, we carried out SILAC-based phosphoproteomic analysis of H358 lung cancer cells chronically exposed to cigarette smoke. We identified 1,812 phosphosites, of which 278 phosphosites were hyperphosphorylated (≥ 3-fold) in H358 cells chronically exposed to cigarette smoke. Our data revealed hyperphosphorylation of S560 within the conserved kinase domain of PAK6. Activation of PAK6 is associated with various processes in cancer including metastasis. Mechanistic studies revealed that inhibition of PAK6 led to reduction in cell proliferation, migration and invasion of the cigarette smoke treated cells. Further, siRNA mediated silencing of PAK6 resulted in decreased invasive abilities in a panel of non-small cell lung cancer (NSCLC) cells. Consistently, mice bearing tumor xenograft showed reduced tumor growth upon treatment with PF-3758309 (group II PAK inhibitor). Immunohistochemical analysis revealed overexpression of PAK6 in 66.6% (52/78) of NSCLC cases in tissue microarrays. Taken together, our study indicates that PAK6 is a promising novel therapeutic target for NSCLC, especially in smokers.

Surface Engineered Protein Nanoparticles With Hyaluronic Acid Based Multilayers For Targeted Delivery Of Anticancer Agents.

Layer-by-layer (LbL) technique was employed to modify the surface of doxorubicin (Dox)-loaded bovine serum albumin (BSA) nanoparticles using hyaluronic acid (HA) to enable targeted delivery to overexpressed CD44 receptors in metastatic breast cancer cells. LbL technique offers a versatile approach to modify the surface of colloidal nanoparticles without any covalent modification. Dox-loaded BSA (Dox Ab) nanoparticles optimized for their size, zeta potential, and drug encapsulation efficiency were prepared by modified desolvation technique. The cellular uptake and cytotoxicity of the LbL coated Dox Ab nanoparticles were analyzed in CD44 overexpressing breast cancer cell line MDA-MB-231. Nanoparticles with HA as the final layer (Dox Ab HA) showed maximum cellular uptake in MDA-MB-231 cells owing to the CD44 receptor-mediated endocytosis and hence, exhibited more cytotoxicity as compared to free Dox. Further, luciferase-transfected MDA-MB-231 cells were used to induce tumor in BALB/c female nude mice to enable whole body tumor imaging. The mice were imaged before and after Dox treatment to visualize the tumor growth. The in vivo biodistribution of Dox Ab HA nanoparticles in nude mice showed maximum accumulation in tumor, and importantly, better tumor reduction in comparison with free Dox, thus paving the way for improved drug delivery into tumors.

Role of the Drug Transporter ABCC3 in Breast Cancer Chemoresistance.

Increased expression of ABC-family of transporters is associated with chemotherapy failure. Although the drug transporters ABCG2, ABCB1 and ABCC1 have been majorly implicated in cancer drug resistance, recent studies have associated ABCC3 with multi drug resistance and poor clinical response. In this study, we have examined the expression of ABCC3 in breast cancers and studied its role in drug resistance and stemness of breast cancer cells in comparison with the more studied ABCC1. We observed that similar to ABCC1, the transcripts levels of ABCC3 was significantly high in breast cancers compared to adjacent normal tissue. Importantly, expression of both transporters was further increased in chemotherapy treated patient samples. Consistent with this, we observed that treatment of breast cancer cell lines with anti-cancer agents increased their mRNA levels of both ABCC1 and ABCC3. Further, similar to knockdown of ABCC1, knockdown of ABCC3 also significantly increased the retention of chemotherapeutic drugs in breast cancer cells and rendered them more chemo-sensitive. Interestingly, ABCC1 and ABCC3 knockdown cells also showed reduction in the expression of stemness genes, while ABCC3 knockdown additionally led to a reduction in the CD44high/CD24low breast cancer stem-like subpopulation. Consistent with this, their ability to form primary tumours was compromised. Importantly, down-modulation of ABCC3 rendered these cells increasingly susceptible to doxorubicin in xenograft mice models in vivo. Thus, our study highlights the importance of ABCC3 transporters in drug resistance to chemotherapy in the context of breast cancer. Further, these results suggest that combinatorial inhibition of these transporters together with standard chemotherapy can reduce therapy-induced resistance in breast cancer.

Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a Comprehensive in Vitro Model for Breast Cancer Metastasis.

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal-based xenograft models are expensive and time-consuming, conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment in vitro to study breast cancer metastasis. Porous poly(ε-caprolactone) (PCL) scaffolds of modulus 7.0 ± 0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis, viz., initiation, progression, and the site-specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere formation efficiency in vitro and increased lung metastasis in vivo. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell-cell and cell-matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB, and HIF1 signaling pathways-all of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the in vivo tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in vitro model to investigate the manifold mechanisms of breast cancer metastasis.

Effects of Withania somnifera and Tinospora cordifolia extracts on the side population phenotype of human epithelial cancer cells: toward targeting multidrug resistance in cancer.

Recent reports suggest the existence of a subpopulation of stem-like cancer cells, termed as cancer stem cells (CSCs), which bear functional and phenotypic resemblance with the adult, tissue-resident stem cells. Side population (SP) assay based on differential efflux of Hoechst 33342 has been effectively used for the isolation of CSCs. The drug resistance properties of SP cells are typically due to the increased expression of ABC transporters leading to drug efflux. Conventionally used chemotherapeutic drugs may often leads to an enrichment of SP, revealing their inability to target the drug-resistant SP and CSCs. Thus, identification of agents that can reduce the SP phenotype is currently in vogue in cancer therapeutics. Withania somnifera (WS) and Tinospora cordifolia (TC) have been used in Ayurveda for treating various diseases, including cancer. In the current study, we have investigated the effects of ethanolic (ET) extracts of WS and TC on the cancer SP phenotype. Interestingly, we found significant decrease in SP on treatment with TC-ET, but not with WS-ET. The SP-inhibitory TC-ET was further fractionated into petroleum ether (TC-PET), dichloromethane (TC-DCM), and n-butyl alcohol (TC-nBT) fractions using bioactivity-guided fractionation. Our data revealed that TC-PET and TC-DCM, but not TC-nBT, significantly inhibited SP in a dose-dependent manner. Furthermore, flow cytometry-based functional assays revealed that TC-PET and TC-DCM significantly inhibited ABC-B1 and ABC-G2 transporters and sensitized cancer cells toward chemotherapeutic drug-mediated cytotoxicity. Thus, the TC-PET and TC-DCM may harbor phytochemicals with the potential to reverse the drug-resistant phenotype, thus improving the efficacy of cancer chemotherapy.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog.

BACKGROUND: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. METHODS: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro- and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. RESULTS: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition (EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NFκB promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NFκB pathway whereas Bmi1 knockdown reduced the expression of NFκB target genes, suggesting that Bmi1 might regulate Nanog expression through the NFκB pathway. CONCLUSIONS: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NFκB pathway.

Coordinate hyperactivation of Notch1 and Ras/MAPK pathways correlates with poor patient survival: novel therapeutic strategy for aggressive breast cancers.

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1(high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined inhibition also abrogated sphere-forming potential, and depleted the putative cancer stem-like cell subpopulation. Most importantly, combinatorial inhibition of Notch1 and Ras/MAPK pathways completely blocked tumor growth in a panel of breast cancer xenografts, including the TNBCs. Thus, our study identifies coordinate hyperactivation of Notch1 and Ras/MAPK pathways as novel biomarkers for poor breast cancer outcome. Furthermore, based on our preclinical data, we propose combinatorial targeting of these two pathways as a treatment strategy for highly aggressive breast cancers, particularly the TNBCs that currently lack any targeted therapeutic module.

Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells.

INTRODUCTION: Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed 'mammospheres'. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression. METHODS: Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. RESULTS: In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser116 phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser116 phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116 residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser116 phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo. CONCLUSIONS: Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.