Sentinel lymph node risk prognostication in primary cutaneous melanoma through tissue-based profiling, potentially redefining the need for sentinel lymph node biopsy.

PURPOSE OF REVIEW: The role of Sentinel Lymph Node Biopsy (SLNB) is pivotal in the contemporary staging of cutaneous melanoma. In this review, we examine advanced molecular testing platforms like gene expression profiling (GEP) and immunohistochemistry (IHC) as tools for predicting the prognosis of sentinel lymph nodes. We compare these innovative approaches with traditional staging assessments. Additionally, we delve into the shared genetic and protein markers between GEP and IHC tests and their relevance to melanoma biology, exploring their prognostic and predictive characteristics. Finally, we assess alternative methods to potentially obviate the need for SLNB altogether. RECENT FINDINGS: Progress in adjuvant melanoma therapy has diminished the necessity of Sentinel Lymph Node Biopsy (SLNB) while underscoring the importance of accurately identifying high-risk stage I and II melanoma patients who may benefit from additional anti-tumor interventions. The clinical application of testing through gene expression profiling (GEP) or immunohistochemistry (IHC) is gaining traction, with platforms such as DecisionDx, Merlin Assay (CP-GEP), MelaGenix GEP, and Immunoprint coming into play. Currently, extensive validation studies are in progress to incorporate routine molecular testing into clinical practice. However, due to significant methodological limitations, widespread clinical adoption of tissue-based molecular testing remains elusive at present. SUMMARY: While various tissue-based molecular testing platforms have the potential to stratify the risk of sentinel lymph node positivity (SLNP), most suffer from significant methodological deficiencies, including limited sample size, lack of prospective validation, and limited correlation with established clinicopathological variables. Furthermore, the genes and proteins identified by individual gene expression profiling (GEP) or immunohistochemistry (IHC) tests exhibit minimal overlap, even when considering the most well-established melanoma mutations. However, there is hope that the ongoing prospective trial for the Merlin Assay may safely reduce the necessity for SLNB procedures if successful. Additionally, the MelaGenix GEP and Immunoprint tests could prove valuable in identifying high-risk stage I-II melanoma patients and potentially guiding their selection for adjuvant therapy, thus potentially reducing the need for SLNB. Due to the diverse study designs employed, effective comparisons between GEP or IHC tests are challenging, and to date, there is no study directly comparing the clinical utility of these respective GEP or IHC tests.

C-reactive protein flare predicts response to checkpoint inhibitor treatment in melanoma.

BACKGROUND: The treatment of melanoma has been revolutionized by the use of immune checkpoint inhibition (ICI), but many patients do not benefit. Furthermore, immune-related adverse events may occur during therapy. A predictive biomarker is needed to reliably identify patients benefitting. In lung, renal cell and bladder cancer early C-reactive protein (CRP) kinetics were shown to be a predictive biomarker for ICI. OBJECTIVE: Here, we investigate early CRP kinetics as predictive biomarker for ICI in melanoma patients. METHODS: Two independent prospectively collected cohorts were analysed: Cohort 1 (n = 87) with advanced and Cohort 2 (n = 99) with completely resected melanoma. Patients were stratified by in the dynamics of CRP after ICI initiation: A doubling of baseline CRP within 30 days followed by at least a 30% drop within 3 months was classified as a CRP flare. If no doubling of CRP was reported, but a 30% drop within 3 months, patients were classified as CRP responders and all others as CRP non-responders. Analysed factors included clinical characteristics like S100B and LDH. Median follow-up was 1.5 and 1.7 years for Cohorts 1 and 2. RESULTS: In Cohort 1 CRP flare (n = 12), CRP responders (n = 43) and CRP non-responders (n = 32) with a progression-free survival (PFS) of 0.7, 0.6 and 0.2 years (p = 0.017) and an overall survival (OS) of 2.2, 1.5 and 1.0 years (p = 0.014), respectively. Multivariable Cox analysis showed an independent risk reduction of progression for CRP responders by 62% compared to CRP non-responders (p = 0.001). In Cohort 2 CRP flare (n = 13), CRP responders (n = 70) and CRP non-responders (n = 16) the log-rank analysis showed a significant difference between OS and recurrence-free survival (RFS) curves (p = 0.046 and p = 0.049). CONCLUSION: Early CRP kinetics could indicate a response to ICI with improved OS and RFS/PFS. CRP flare and CRP response indicating significantly improved outcomes compared to CRP non-responders.

Circulating tumor cells as liquid biopsy markers in cancer patients.

Over the past decade, novel methods for enrichment and identification of cancer cells circulating in the blood have been established. Blood-based detection of cancer cells and other tumor-associated products can be summarized under the term of Liquid Biopsy. Circulating tumor cells (CTCs) have been used for diagnosis, risk stratification and treatment selection as well as treatment monitoring in several studies over the past years, thus representing a valuable biomarker for cancer patients. A plethora of methods to enrich, detect and analyze CTCs has been established. In contrast to other liquid biopsy analytes (e.g. ctDNA), CTCs represent a viable analyte that provides a unique opportunity to understand the underlaying biology of cancer and the metastatic cascade on the molecular level. In this review, we provide an overview on the current methods used for enrichment, detection, molecular and functional characterization of CTCs.

All Three AKT Isoforms Can Upregulate Oxygen Metabolism and Lactate Production in Human Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC), the main pathological type of liver cancer, is related to risk factors such as viral hepatitis, alcohol intake, and non-alcoholic fatty liver disease (NAFLD). The constitutive activation of the PI3K/AKT signaling pathway is common in HCC and has essential involvement in tumor progression. The serine/threonine kinase AKT has several downstream substrates, which have been implicated in the regulation of cellular metabolism. However, the contribution of each of the three AKT isoforms, i.e., AKT1, AKT2 and AKT3, to HCC metabolism has not been comprehensively investigated. In this study, we analyzed the functional role of AKT1, AKT2 and AKT3 in HCC metabolism. The overexpression of activated AKT1, AKT2 and AKT3 isoforms in the human HCC cell lines Hep3B and Huh7 resulted in higher oxygen consumption rate (OCR), ATP production, maximal respiration and spare respiratory capacity in comparison to vector-transduced cells. Vice versa, lentiviral vector-mediated knockdowns of each AKT isoform reduced OCR in both cell lines. Reduced OCR rates observed in the three AKT isoform knockdowns were associated with reduced extracellular acidification rates (ECAR) and reduced lactate production in both analyzed cell lines. Mechanistically, the downregulation of OCR by AKT isoform knockdowns correlated with an increased phosphorylation of the pyruvate dehydrogenase on Ser232, which negatively regulates the activity of this crucial gatekeeper of mitochondrial respiration. In summary, our data indicate that each of the three AKT isoforms is able to upregulate OCR, ECAR and lactate production independently of each other in human HCC cells through the regulation of the pyruvate dehydrogenase.

K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3.

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.

Clinical applications of circulating tumor cells in patients with solid tumors.

The concept of liquid biopsy analysis has been established more than a decade ago. Since the establishment of the term, tremendous advances have been achieved and plenty of methods as well as analytes have been investigated in basic research as well in clinical trials. Liquid biopsy refers to a body fluid-based biopsy that is minimal-invasive, and most importantly, allows dense monitoring of tumor responses by sequential blood sampling. Blood is the most important analyte for liquid biopsy analyses, providing an easily accessible source for a plethora of cells, cell-derived products, free nucleic acids, proteins as well as vesicles. More than 12,000 publications are listed in PubMed as of today including the term liquid biopsy. In this manuscript, we critically review the current implications of liquid biopsy, with special focus on circulating tumor cells, and describe the hurdles that need to be addressed before liquid biopsy can be implemented in clinical standard of care guidelines.

[Liquid Biopsy - A new diagnostic concept in oncology]. / Liquid Biopsy ­ Ein neues diagnostisches Konzept in der Onkologie.

The analysis of tumor cells circulating in the blood or of products of tumor cells circulating in other body fluids has gained increasing attention in recent years and is summarized under the term liquid biopsy (LB). LB includes the analysis of circulating tumor cells, cell-free circulating tumor-associated nucleic acids, extracellular vesicles, proteins, or other products that are released into the peripheral bloodstream by the primary or metastatic tumor. For a huge number of solid tumor entities, LB has already been successfully applied in preclinical and clinical studies for the detection, risk stratification, treatment monitoring and relapse detection. LB provides valuable real-time information on tumor cell development, therapeutic targets, and mechanisms of therapy resistance using a non-invasive peripheral blood test. In this article, the most important LB analytes and the current state of research are presented. In addition, the remaining obstacles and the diverse efforts to implement LB in clinical routine are critically discussed.

Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis.

Introduction: The PI3K/AKT pathway is activated in 43-70% of breast cancer (BC)-patients and promotes the metastatic potential of BC cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for triple-negative breast cancer (TNBC)-patients with brain metastases. Methods: The impact of AKT1-knockout (AKT1_KO) and AKT-inhibition using Ipatasertib on MDA-MB-231 BR cells was assessed using in vitro cell proliferation and migration assays. AKT1-knockout in MDA-MB-231BR cells was performed using CRISPR/Cas9. The effect of AKT1-knockout on radiosensitivity of MDA-MB-231BR cell lines was determined via colony formation assays after cell irradiation. To detect genomic variants in AKT1_KO MDA-MB-231BR cells, whole-genome sequencing (WGS) was performed. Results: Pharmacological inhibition of AKT with the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability but did not impact cell migration. Moreover, only MDA-MB-231BR cells were sensitized following Ipatasertib-treatment. Furthermore, specific AKT1-knockout in MDA-MB-231BR showed reduced cell viability in comparison to control cells, with significant effect in one of two analyzed clones. Unexpectedly, AKT1 knockout led to increased cell migration and clonogenic potential in both AKT1_KO clones. RNAseq-analysis revealed the deregulation of CTSO, CYBB, GPR68, CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN leading to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 knockout in MDA-MB-231BR. Discussion: Collectively, our results demonstrate that Ipatasertib leads to radiosensitization and reduced cell proliferation of MDA-MB-231BR. AKT1-inhibition showed altered gene expression profile leading to modified cell migration, clonogenic survival and radioresistance in MDA-MB-231BR. We conclude, that AKT1-inhibition in combination with radiotherapy contribute to novel treatment strategies for breast cancer brain metastases.

[Liquid Biopsy - A new diagnostic concept in oncology]. / Liquid Biopsy ­ Ein neues diagnostisches Konzept in der Onkologie.

The analysis of tumor cells circulating in the blood or of products of tumor cells circulating in other body fluids has gained increasing attention in recent years and is summarized under the term liquid biopsy (LB). LB includes the analysis of circulating tumor cells, cell-free circulating tumor-associated nucleic acids, extracellular vesicles, proteins, or other products that are released into the peripheral bloodstream by the primary or metastatic tumor. For a huge number of solid tumor entities, LB has already been successfully applied in preclinical and clinical studies for the detection, risk stratification, treatment monitoring and relapse detection. LB provides valuable real-time information on tumor cell development, therapeutic targets, and mechanisms of therapy resistance using a non-invasive peripheral blood test. In this article, the most important LB analytes and the current state of research are presented. In addition, the remaining obstacles and the diverse efforts to implement LB in clinical routine are critically discussed.

The Role of PI3K/AKT/mTOR Signaling in Hepatocellular Carcinoma Metabolism.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths in the world. Metabolic reprogramming is considered a new hallmark of cancer, but it remains unclearly described in HCC. The dysregulation of the PI3K/AKT/mTOR signaling pathway is common in HCC and is, therefore, a topic of further research and the concern of developing a novel target for liver cancer therapy. In this review, we illustrate mechanisms by which this signaling network is accountable for regulating HCC cellular metabolism, including glucose metabolism, lipid metabolism, amino acid metabolism, pyrimidine metabolism, and oxidative metabolism, and summarize the ongoing clinical trials based on the inhibition of the PI3K/AKT/mTOR pathway in HCC.

TIGIT blockade repolarizes AML-associated TIGIT+ M2 macrophages to an M1 phenotype and increases CD47-mediated phagocytosis.

BACKGROUND: Leukemia-associated macrophages (LAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype, function, and plasticity of these cells. The present study provides an extensive characterization of macrophages in patients with acute myeloid leukemia (AML). METHODS: The phenotype and expression of coregulatory markers were assessed on bone marrow (BM)-derived LAM populations, using multiparametric flow cytometry. BM and blood aspirates were obtained from patients with newly diagnosed acute myeloid leukemia (pAML, n=59), patients in long-term remission (lrAML, n=8), patients with relapsed acute myeloid leukemia (rAML, n=7) and monocyte-derived macrophages of the blood from healthy donors (HD, n=17). LAM subpopulations were correlated with clinical parameters. Using a blocking anti-T-cell immunoreceptor with Ig and ITIM domains (TIGIT) antibody or mouse IgG2α isotype control, we investigated polarization, secretion of cytokines, and phagocytosis on LAMs and healthy monocyte-derived macrophages in vitro. RESULTS: In pAML and rAML, M1 LAMs were reduced and the predominant macrophage population consisted of immunosuppressive M2 LAMs defined by expression of CD163, CD204, CD206, and CD86. M2 LAMs in active AML highly expressed inhibitory receptors such as TIGIT, T-cell immunoglobulin and mucin-domain containing-3 protein (TIM-3), and lymphocyte-activation gene 3 (LAG-3). High expression of CD163 was associated with a poor overall survival (OS). In addition, increased frequencies of TIGIT+ M2 LAMs were associated with an intermediate or adverse risk according to the European Leukemia Network criteria and the FLT3 ITD mutation. In vitro blockade of TIGIT shifted the polarization of primary LAMs or peripheral blood-derived M2 macrophages toward the M1 phenotype and increased secretion of M1-associated cytokines and chemokines. Moreover, the blockade of TIGIT augmented the anti-CD47-mediated phagocytosis of AML cell lines and primary AML cells. CONCLUSION: Our findings suggest that immunosuppressive TIGIT+ M2 LAMs can be redirected into an efficient effector population that may be of direct clinical relevance in the near future.

AKT Isoforms as a Target in Cancer and Immunotherapy.

Over the past years, targeted therapies have received tremendous attention in cancer therapy. One of the most frequently targeted pathways is the PI3K/AKT/mTOR signaling pathway that regulates crucial cellular processes including proliferation, survival, and migration. In a wide variety of cancer entities, the PI3K/AKT/mTOR signaling pathway was found to be a critical driver of disease progression, indicating a noteworthy target in cancer therapy. This chapter focuses on targeted therapies against AKT, which is a key enzyme within the PI3K/AKT/mTOR pathway. Although the three different isoforms of AKT, namely AKT1, AKT2, and AKT3, have a high homology, the isoforms exhibit different biological functions. Recently, direct inhibitors against all AKT isoforms as well as selective inhibitors against specific AKT isoforms have been extensively investigated in preclinical work as well as in clinical trials to attenuate proliferation of cancer cells. While no AKT inhibitor has been approved by the FDA for cancer therapy to date, AKT still plays a crucial role in a variety of treatment strategies including immune checkpoint inhibition. In this chapter, we summarize the status of AKT inhibitors either targeting all or specific AKT isoforms. Furthermore, we explain the role of AKT signaling in direct inhibition of tumor cell growth as well as in immune cells and immune checkpoint inhibition.

The role of sphingosine-1-phosphate in bone remodeling and osteoporosis.

Osteoporosis is a systemic bone disease that affects more than 200 million people worldwide and is caused by the disruption of the equilibrium between osteoclastic bone resorption and osteoblastic bone formation. Sphingosine-1-phosphate (S1P) is a natural, bioactive sphingolipid that has been shown to play a major role in cardiovascular and immunological pathologies by regulating biological and cellular processes, including migration, differentiation, proliferation and survival. Recent studies also suggest a central role for S1P in bone diseases, including osteoporosis; however, the effects of S1P, particularly in bone metabolism, remain to be further elucidated. In this review, we summarize the available literature on the role of S1P in bone metabolism with a focus on osteoporosis. On the cellular level, S1P acts as an osteoclast-osteoblast coupling factor to promote osteoblast proliferation and bone formation. Moreover, the recruitment of osteoclast precursors to resorption sites is regulated by the interplay of S1P gradients and S1P receptor expression. From a clinical perspective, increasing evidence suggests that systemically elevated S1P blood levels may serve as an independent risk factor for osteoporosis-related fractures. Taken together, S1P signaling is a potential therapeutic target and may serve as a novel biomarker in patients with systemic bone disease.

Combined Targeting of AKT and mTOR Inhibits Tumor Formation of EpCAM+ and CD90+ Human Hepatocellular Carcinoma Cells in an Orthotopic Mouse Model.

The epithelial cell adhesion molecule (EpCAM) and Thy-1 cell surface antigen (CD90) have been implicated as cancer stem cell (CSC) markers in hepatocellular carcinoma (HCC). Expression of EpCAM and CD90 on HCC cells is associated with increased tumorigenicity, metastasis and poor prognosis. In this study, we demonstrate that combined treatment with AKT and mTOR inhibitors-i.e., MK2206 and RAD001-results in a synergistic reduction in proliferation of EpCAM+ and CD90+ HCC cells cultured either as adherent cells or as tumoroids in vitro. In addition, tumor growth was reduced by combined treatment with AKT and mTOR inhibitors in an orthotopic xenograft mouse model of an EpCAM+ HCC cell line (Huh7) and primary patient-derived EpCAM+ HCC cells (HCC1) as well as a CD90+ HCC-related cell line (SK-HEP1) in vivo. However, during AKT/mTOR treatment, outgrowth of therapy-resistant tumors was observed in all mice analyzed within a few weeks. Resistance was associated in most cases with restoration of AKT signaling in the tumors, intrahepatic metastases and distant metastases. In addition, an upregulation of the p38 MAPK pathway was identified in the AKT/mTOR inhibitor-resistant tumor cells by kinome profiling. The development of resistant cells during AKT/mTOR therapy was further analyzed by red-green-blue (RGB) marking of HCC cells, which revealed an outgrowth of a large number of Huh7 cells over a period of 6 months. In summary, our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of EpCAM+ as well as CD90+ HCC cells in vitro. However, the fast development of large numbers of resistant clones under AKT/mTOR therapy observed in vitro and in the orthotopic xenotransplantation mouse model in vivo strongly suggests that this therapy alone will not be sufficient to eliminate EpCAM+ or CD90+ cancer stem cells from HCC patients.

MicroRNAs: Emerging Regulators of Metastatic Bone Disease in Breast Cancer.

Bone metastasis is a frequent complication in patients with advanced breast cancer. Once in the bone, cancer cells disrupt the tightly regulated cellular balance within the bone microenvironment, leading to excessive bone destruction and further tumor growth. Physiological and pathological interactions in the bone marrow are mediated by cell-cell contacts and secreted molecules that include soluble proteins as well as RNA molecules. MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally interfere with their target messenger RNA (mRNA) and subsequently reduce protein abundance. Since their discovery, miRNAs have been identified as critical regulators of physiological and pathological processes, including breast cancer and associated metastatic bone disease. Depending on their targets, miRNAs can exhibit pro-tumorigenic or anti-tumorigenic functions and serve as diagnostic and prognostic biomarkers. These properties have encouraged pre-clinical and clinical development programs to investigate miRNAs as biomarkers and therapeutic targets in various diseases, including metastatic cancers. In this review, we discuss the role of miRNAs in metastatic bone disease with a focus on breast cancer and the bone microenvironment and elaborate on their potential use for diagnostic and therapeutic purposes in metastatic bone disease and beyond.

High Serum Levels of Wnt Signaling Antagonist Dickkopf-Related Protein 1 Are Associated with Impaired Overall Survival and Recurrence in Esophageal Cancer Patients.

Dickkopf-related protein 1 (DKK1), an antagonist of the canonical Wnt pathway, has received tremendous attention over the past years as its dysregulation is said to be critically involved in a wide variety of gastrointestinal cancers. However, the potential clinical implications of DKK1 remain poorly understood. Although multimodal treatment options have been implemented over the past years, esophageal cancer (EC) patients still suffer from poor five-year overall survival rates ranging from 15% to 25%. Especially prognostic factors and biomarkers for risk stratification are lacking to choose the most beneficial treatment out of the emerging landscape of different treatment options. In this study, we analyzed the serum DKK1 (S-DKK1) levels of 91 EC patients prior to surgery in a single center study at the University Medical Center Hamburg-Eppendorf by enzyme-linked immunosorbent assay. High levels of S-DKK1 could be especially observed in patients suffering from esophageal adenocarcinoma which may promote the hypothesis of a crucial role of DKK1 in inflammation. S-DKK1 levels of ≥5800 pg/mL were shown to be associated with unfavorable five-year survival rates and the presence of CTCs. Interestingly, significantly lower S-DKK1 levels were detected in patients after neoadjuvant treatment, implying that S-DKK1 may serve as a useful biomarker for treatment monitoring. Multivariate analysis identified S-DKK1 as an independent prognostic marker with respect to overall survival in EC patients with a hazard ratio of 2.23. In conclusion, our data implicate a negative prognostic role of DKK1 with respect to the clinical outcome in EC patients. Further prospective studies should be conducted to implement S-DKK1 into the clinical routine for risk stratification and treatment monitoring.

Circulating tumor cells as a promising target for individualized drug susceptibility tests in cancer therapy.

Circulating tumor cells (CTCs) play a crucial role in metastasis and became an emerging topic in today's cancer research. In addition, the analysis of CTCs in liquid biopsies will be a valuable tool for prognosis prediction and real time therapy monitoring. The characterization of CTCs may open up a new field of treatment strategy to prevent metastasis or maintain a stable disease. In 2013, the first cell cultures of CTCs have been established in vitro. Additionally, functional studies have been successfully performed over the last years. Meanwhile, more than 300 short-term CTC cultures and 42 long-term CTC cultures from a variety of tumor entities have been described. More than 45 inhibitors have already been tested for their efficacy to target CTCs in several studies in vitro as well as in xenograft mouse models in vivo. Here, we summarize the currently available data of these inhibition experiments and their effects in targeting CTCs. The results suggest that CTCs may be useful for individualized drug susceptibility testing.

Combined Targeting of AKT and mTOR Synergistically Inhibits Formation of Primary Colorectal Carcinoma Tumouroids In Vitro: A 3D Tumour Model for Pre-therapeutic Drug Screening.

BACKGROUND: Pre-therapeutic analysis of three-dimensional spheroid cultures of primary tumour samples is a promising approach of assessing susceptibility to potential treatment. The phosphatidylinositol-3-kinase/AKT serine/threonine kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is frequently activated in colorectal cancer (CRC). In previous work, we showed combined inhibition of AKT and mTOR to be highly synergistic in cell lines from patients with hepatocellular carcinoma and cholangiocarcinoma in vitro as well as in vivo in murine xenograft tumour models. MATERIALS AND METHODS: Patient-derived xenograft colorectal carcinoma cell lines HROC80 T1 M1, HROC147 T0 M1, HROC147Met, HROC277 T0 M1 and HROC277Met2 were treated with AKT inhibitor MK2206, mTOR inhibitor RAD001 or the combination of both drugs. The sensitivity of these cell lines to inhibition was evaluated by calculation of combinatory indices after bromodeoxyuridine assays and analysis of the respective pathways by western blotting. Furthermore, the dual inhibition of AKT and mTOR was confirmed in vivo in a xenograft mouse model. Additionally, primary CRC samples of four patients were embedded in a three-dimensional matrix and the sensitivity of these samples was analyzed by measurement of the spheroid area. RESULTS: In this study, we demonstrate that combined treatment with MK2206 and RAD001 resulted in strong synergistic effects on growth of several primary CRC cell lines and reduced the growth of a patient-derived CRC xenograft in a xenotransplantation mouse model in vivo. Interestingly, the response to treatment varied between cell lines derived from the primary lesion and a liver metastasis of the same patient. In addition, combined treatment with AKT and mTOR inhibitors resulted in a synergistic inhibition of tumouroid growth in all four of the primary patient samples, analyzed in a three-dimensional spheroid model in vitro. CONCLUSION: Our data demonstrate that combined treatment with AKT and mTOR inhibitors exhibits synergistic effects on proliferation of cell lines and primary tumour cells from patients with CRC and may be a promising approach for the treatment of CRC.

Interleukins as Mediators of the Tumor Cell-Bone Cell Crosstalk during the Initiation of Breast Cancer Bone Metastasis.

Patients with advanced breast cancer are at high risk of developing bone metastasis. Despite treatment advances for primary breast cancer, metastatic bone disease remains incurable with a low relative survival. Hence, new therapeutic approaches are required to improve survival and treatment outcome for these patients. Bone is among the most frequent sites of metastasis in breast cancer. Once in the bone, disseminated tumor cells can acquire a dormant state and remain quiescent until they resume growth, resulting in overt metastasis. At this stage the disease is characterized by excessive, osteoclast-mediated osteolysis. Cells of the bone microenvironment including osteoclasts, osteoblasts and endothelial cells contribute to the initiation and progression of breast cancer bone metastasis. Direct cell-to-cell contact as well as soluble factors regulate the crosstalk between disseminated breast cancer cells and bone cells. In this complex signaling network interleukins (ILs) have been identified as key regulators since both, cancer cells and bone cells secrete ILs and express corresponding receptors. ILs regulate differentiation and function of bone cells, with several ILs being reported to act pro-osteoclastogenic. Consistently, the expression level of ILs (e.g., in serum) has been associated with poor prognosis in breast cancer. In this review we discuss the role of the most extensively investigated ILs during the establishment of breast cancer bone metastasis and highlight their potential as therapeutic targets in preventing metastatic outgrowth in bone.

Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFß/CTGF Axis.

Bone metastases frequently occur in breast cancer patients and lack appropriate treatment options. Hence, understanding the molecular mechanisms involved in the multistep process of breast cancer bone metastasis and tumor-induced osteolysis is of paramount interest. The serine/threonine kinase AKT plays a crucial role in breast cancer bone metastasis but the effect of individual AKT isoforms remains unclear. Therefore, AKT isoform-specific knockdowns were generated on the bone-seeking MDA-MB-231 BO subline and the effect on proliferation, migration, invasion, and chemotaxis was analyzed by live-cell imaging. Kinome profiling and Western blot analysis of the TGFß/CTGF axis were conducted and metastasis was evaluated by intracardiac inoculation of tumor cells into NOD scid gamma (NSG) mice. MDA-MB-231 BO cells exhibited an elevated AKT3 kinase activity in vitro and responded to combined treatment with AKT- and mTOR-inhibitors. Knockdown of AKT3 significantly increased migration, invasion, and chemotaxis in vitro and metastasis to bone but did not significantly enhance osteolysis. Furthermore, knockdown of AKT3 increased the activity and phosphorylation of pro-metastatic HER2 and DDR1/2 but lowered protein levels of CTGF after TGFß-stimulation, an axis involved in tumor-induced osteolysis. We demonstrated that AKT3 plays a crucial role in bone-seeking breast cancer cells by promoting metastatic potential without facilitating tumor-induced osteolysis.