Targeting SMAD-Dependent Signaling: Considerations in Epithelial and Mesenchymal Solid Tumors.

SMADs are the canonical intracellular effector proteins of the TGF-ß (transforming growth factor-ß). SMADs translocate from plasma membrane receptors to the nucleus regulated by many SMAD-interacting proteins through phosphorylation and other post-translational modifications that govern their nucleocytoplasmic shuttling and subsequent transcriptional activity. The signaling pathway of TGF-ß/SMAD exhibits both tumor-suppressing and tumor-promoting phenotypes in epithelial-derived solid tumors. Collectively, the pleiotropic nature of TGF-ß/SMAD signaling presents significant challenges for the development of effective cancer therapies. Here, we review preclinical studies that evaluate the efficacy of inhibitors targeting major SMAD-regulating and/or -interacting proteins, particularly enzymes that may play important roles in epithelial or mesenchymal compartments within solid tumors.

Multidimensional screening of pancreatic cancer spheroids reveals vulnerabilities in mitotic and cell-matrix adhesion signaling that associate with metastatic progression and decreased patient survival.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, with a median survival of less than 12 months and a 5-year survival of less than 10 %. Here, we have established an image-based screening pipeline for quantifying single PDAC spheroid dynamics in genetically and phenotypically diverse PDAC cell models. Wild-type KRas PDAC cells formed tight/compact spheroids - compaction of these structures was completely blocked by cytoplasmic dynein and focal adhesion kinase (FAK) inhibitors. In contrast, PDAC cells containing mutant KRas formed loosely aggregated spheroids that grew significantly slower following inhibition of polo-like kinase 1 (PLK1) or focal adhesion kinase (FAK). Independent of genetic background, multicellular PDAC-mesenchymal stromal cell (MSC) spheroids self-organized into structures with an MSC-dominant core. The inclusion of MSCs into wild-type KRas PDAC spheroids modestly affected their compaction; however, MSCs significantly increased the compaction and growth of mutant KRas PDAC spheroids. Notably, exogenous collagen 1 potentiated PANC1 spheroid compaction while ITGA1 knockdown in PANC1 cells blocked MSC-induced PANC1 spheroid compaction. In agreement with a role for collagen-based integrin adhesion complexes in stromal cell-induced PDAC phenotypes, we also discovered that MSC-induced PANC1 spheroid growth was completely blocked by the ITGB1 immunoneutralizing antibody mAb13. Finally, multiplexed single-cell immunohistochemical analysis of a 25 patient PDAC tissue microarray revealed a relationship between decreased variance in Spearman r correlation for ITGA1 and PLK1 expression within the tumor cell compartment of PDAC in patients with advanced disease stage, and elevated expression of both ITGA1 and PLK1 in PDAC was found to be associated with decreased patient survival. Taken together, this work uncovers new therapeutic vulnerabilities in PDAC that are relevant to the progression of this stromal cell-rich malignancy and which may reveal strategies for improving patient outcomes.

Patient-Derived Organoids as Therapy Screening Platforms in Cancer Patients.

Patient-derived organoids (PDOs) developed ex vivo and in vitro are increasingly used for therapeutic screening. They provide a more physiologically relevant model for drug discovery and development compared to traditional cell lines. However, several challenges remain to be addressed to fully realize the potential of PDOs in therapeutic screening. This paper summarizes recent advancements in PDO development and the enhancement of PDO culture models. This is achieved by leveraging materials engineering and microfabrication technologies, including organs-on-a-chip and droplet microfluidics. Additionally, this work discusses the application of PDOs in therapy screening to meet diverse requirements and overcome bottlenecks in cancer treatment. Furthermore, this work introduces tools for data processing and analysis of organoids, along with their microenvironment. These tools aim to achieve enhanced readouts. Finally, this work explores the challenges and future perspectives of using PDOs in drug development and personalized screening for cancer patients.

Fibronectin, DHPS and SLC3A2 Signaling Cooperate to Control Tumor Spheroid Growth, Subcellular eIF5A1/2 Distribution and CDK4/6 Inhibitor Resistance.

Extracellular matrix (ECM) protein expression/deposition within and stiffening of the breast cancer microenvironment facilitates disease progression and correlates with poor patient survival. However, the mechanisms by which ECM components control tumorigenic behaviors and responses to therapeutic intervention remain poorly understood. Fibronectin (FN) is a major ECM protein controlling multiple processes. In this regard, we previously reported that DHPS-dependent hypusination of eIF5A1/2 is necessary for fibronectin-mediated breast cancer metastasis and epithelial to mesenchymal transition (EMT). Here, we explored the clinical significance of an interactome generated using hypusination pathway components and markers of intratumoral heterogeneity. Solute carrier 3A2 (SLC3A2 or CD98hc) stood out as an indicator of poor overall survival among patients with basal-like breast cancers that express elevated levels of DHPS. We subsequently discovered that blockade of DHPS or SLC3A2 reduced triple negative breast cancer (TNBC) spheroid growth. Interestingly, spheroids stimulated with exogenous fibronectin were less sensitive to inhibition of either DHPS or SLC3A2 - an effect that could be abrogated by dual DHPS/SLC3A2 blockade. We further discovered that a subset of TNBC cells responded to fibronectin by increasing cytoplasmic localization of eIF5A1/2. Notably, these fibronectin-induced subcellular localization phenotypes correlated with a G0/G1 cell cycle arrest. Fibronectin-treated TNBC cells responded to dual DHPS/SLC3A2 blockade by shifting eIF5A1/2 localization back to a nucleus-dominant state, suppressing proliferation and further arresting cells in the G2/M phase of the cell cycle. Finally, we observed that dual DHPS/SLC3A2 inhibition increased the sensitivity of both Rb-negative and -positive TNBC cells to the CDK4/6 inhibitor palbociclib. Taken together, these data identify a previously unrecognized mechanism through which extracellular fibronectin controls cancer cell tumorigenicity by modulating subcellular eIF5A1/2 localization and provides prognostic/therapeutic utility for targeting the cooperative DHPS/SLC3A2 signaling axis to improve breast cancer treatment responses.

Impact of COVID-19 pandemic on rare diseases - A case study on thalassaemia patients in Bangladesh.

OBJECTIVES: Thalassaemia is a life-threatening rare disease, which requires regular blood transfusion and medical care. The information on how thalassaemia patients are affected during the unprecedented COVID-19 crisis is scarce. This study aimed to assess the impact of the COVID-19 pandemic on the blood transfusion and healthcare access of thalassaemia patients at the community level in Bangladesh. METHODS: A cross-sectional study was conducted among thalassaemia patients registered in a community-based 'thalassaemia registry' in Jamapur, Bangladesh. RESULTS: As compared to pre-COVID-19 time, the number of blood transfusions among patients under the thalassaemia registry was significantly reduced during COVID-19 pandemic (190 units versus 81 units). In addition, the median number of red cell transfusions per patient was dropped significantly from 4 units to one unit. Over 80% of patient had no access to healthcare services at all during the early phase of the pandemic. CONCLUSIONS: Emergency response with appropriate mitigative measures must be a priority for addressing an acute shortage of blood supply in situations like COVID-19 pandemic.

A SNAI2-PEAK1-INHBA stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting subpopulations of tumor cells positive for antiapoptotic and stress signaling markers.

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Specifically, we identified that the fibroblastic stroma in HER2-positive breast cancer patient tissue stains positive for both nuclear SNAI2 and cytoplasmic PEAK1. Furthermore, mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells in a PEAK1-dependent manner. Analysis of PEAK1-dependent secreted factors from MSCs revealed INHBA/activin-A as a necessary factor in the conditioned media of PEAK1-expressing MSCs that promotes lapatinib resistance. Single-cell CycIF analysis of MSC-breast cancer cell co-cultures identified enrichment of p-Akthigh/p-gH2AXlow, MCL1high/p-gH2AXlow and GRP78high/VIMhigh breast cancer cell subpopulations by the presence of PEAK1-expressing MSCs and lapatinib treatment. Bioinformatic analyses on a PEAK1-centric stroma-tumor cell gene set and follow-up immunostaining of co-cultures predict targeting antiapoptotic and stress pathways as a means to improve targeted therapy responses and patient outcomes in HER2-positive breast cancer and other stroma-rich malignancies. These data provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA stromal cell axis.

Identification of myosin II as a cripto binding protein and regulator of cripto function in stem cells and tissue regeneration.

Cripto regulates stem cell function in normal and disease contexts via TGFbeta/activin/nodal, PI3K/Akt, MAPK and Wnt signaling. Still, the molecular mechanisms that govern these pleiotropic functions of Cripto remain poorly understood. We performed an unbiased screen for novel Cripto binding proteins using proteomics-based methods, and identified novel proteins including members of myosin II complexes, the actin cytoskeleton, the cellular stress response, and extracellular exosomes. We report that myosin II, and upstream ROCK1/2 activities are required for localization of Cripto to cytoplasm/membrane domains and its subsequent release into the conditioned media fraction of cultured cells. Functionally, we demonstrate that soluble Cripto (one-eyed pinhead in zebrafish) promotes proliferation in mesenchymal stem cells (MSCs) and stem cell-mediated wound healing in the zebrafish caudal fin model of regeneration. Notably, we demonstrate that both Cripto and myosin II inhibitors attenuated regeneration to a similar degree and in a non-additive manner. Taken together, our data present a novel role for myosin II function in regulating subcellular Cripto localization and function in stem cells and an important regulatory mechanism of tissue regeneration. Importantly, these insights may further the development of context-dependent Cripto agonists and antagonists for therapeutic benefit.

Ascending the PEAK1 toward targeting TGFß during cancer progression: Recent advances and future perspectives.

Cancer is the second leading cause of death in the United States. Mortality in patients with solid, epithelial-derived tumors strongly correlates with disease stage and the systemic metastatic load. In such cancers, notable morphological and molecular changes have been attributed to cells as they pass through a continuum of epithelial-mesenchymal transition (EMT) states and many of these changes are essential for metastasis. While cancer metastasis is a complex cascade that is regulated by cell-autonomous and microenvironmental influences, it is well-accepted that understanding and controlling metastatic disease is a viable method for increasing patient survival. In the past 5 years, the novel non-receptor tyrosine kinase PEAK1 has surfaced as a central regulator of tumor progression and metastasis in the context of solid, epithelial cancers. Here, we review this literature with a special focus on our recent work demonstrating that PEAK1 mediates non-canonical pro-tumorigenic TGFß signaling and is an intracellular control point between tumor cells and their extracellular microenvironment. We conclude with a brief discussion of potential applications derived from our current understanding of PEAK1 biology.

Nuclear heterogeneity in conidial populations of Aspergillus flavus.

Aspergillus flavus is a major producer of aflatoxin and an opportunistic pathogen for a wide range of hosts. Understanding genotypic and phenotypic variation within strains of A. flavus is important for controlling disease and reducing aflatoxin contamination. A. flavus is multinucleate and predominantly haploid (n) and homokaryotic. Although cryptic heterokaryosis may occur in nature, it is unclear how nuclei in A. flavus influence genetic heterogeneity and if nuclear condition plays a role in fungal ecology. A. flavus mainly reproduces asexually by producing conidia. In order to observe whether conidia are homokaryotic or heterokaryotic, we labeled nuclei of A. flavus using two different nuclear localized fluorescent reporters. The reporter constructs (pYH2A and pCH2B), encode histones HH2A and HH2B fused at the C terminus with either yellow (EYFP) or cyan (ECFP) fluorescent proteins, respectively. The constructs were transformed into the double auxotrophic strain AFC-1 (-pyrG, -argD) to generate a strain containing each reporter construct. By taking advantage of the nutritional requirement for each strain, we were able to generate fusants between FR36 (-argD) expressing yellow fluorescence, and FR46 (-pyr4) expressing cyan fluorescence. Conidia from fusants between FR36 and FR46 showed three types of fluorescence: only EYFP, only ECFP or both EYFP+ECFP. Conidia containing nuclei expressing EYFP+ECFP were separated by Fluorescence-Activated Cell sorting (FACS) and were found to contain both yellow and cyan fluorescent markers in the same nucleus. Further characterization of conidia having only one nucleus but expressing both EYFP+ECFP fluorescence were found to be diploid (2n). Our findings suggest that A. flavus maintains nuclear heterogeneity in conidial populations.

Identification of a PEAK1/ZEB1 signaling axis during TGFß/fibronectin-induced EMT in breast cancer.

Transforming Growth Factor beta (TGFß) is the archetypal member of the TGFß superfamily of ligands and has pleiotropic functions during normal development, adult tissue homeostasis and pathophysiological processes such as cancer. In epithelial cancers TGFß signaling can either suppress tumor growth or promote metastasis via the induction of a well-characterized epithelial-mesenchymal transition (EMT) program. We recently reported that PEAK1 kinase mediates signaling cross talk between TGFß receptors and integrin/Src/MAPK pathways and functions as a critical molecular regulator of TGFß-induced breast cancer cell proliferation, migration, EMT and metastasis. Here, we examined the breast cancer cell contexts in which TGFß induces both EMT and PEAK1, and discovered this event to be unique to oncogene-transformed mammary epithelial cells and triple-negative breast cancer cells. Using the Cancer BioPortal database, we identified PEAK1 co-expressors across multiple malignancies that are also common to the TGFß response gene signature (TBRS). We then used the ScanSite database to identify predicted protein-protein binding partners of PEAK1 and the PEAK1-TBRS co-expressors. Analysis of the Cytoscape interactome and Babelomics-derived gene ontologies for a novel gene set including PEAK1, CRK, ZEB1, IL11 and COL4A1 enabled us to hypothesize that PEAK1 may be regulating TGFß-induced EMT via its interaction with or regulation of these other genes. In this regard, we have demonstrated that PEAK1 is necessary for TGFß to induce ZEB1-mediated EMT in the context of fibronectin/ITGB3 activation. These studies and future mechanistic studies will pave the way toward identifying the context in which TGFß blockade may significantly improve breast cancer patient outcomes.

Molecular versus conventional methods: clinical evaluation of different methods for the diagnosis of tuberculosis in Bangladesh.

BACKGROUND: The diagnosis of tuberculosis (TB) in developing countries, such as Bangladesh, is based mainly on microscopic detection of acid-fast bacilli in smears from clinical specimens. On the other hand, the detection of TB by polymerase chain reaction (PCR) is quite new in Bangladesh. In this study, we compared the molecular method with the conventional diagnosis procedures, where Lowenstein-Jensen medium culture results have been used as the "gold standard." METHODS: A total of 135 sputum samples were collected from clinically suspected patients with pulmonary TB. A direct smear was made from each sputum specimen and stained by the Ziehl-Neelsen (Z-N) method. The sputum samples were then processed, and the pellet was used for both Z-N (concentration) and auramine O fluorescence staining or resuspended in phosphate buffered saline to inoculate Lowenstein-Jensen medium or processed for PCR detection of Mycobacterium tuberculosis. RESULTS: The direct smear staining yielded 44 (32.6%) sputum samples that were acid-fast positive, which increased after concentration, yielding 60 (44.4%) acid-fast-positive samples. Fluorescence microscopy using auramine O staining further increased the number of positive samples to 67 (49.6%). The biochemical tests showed 75 (55.6%) sputum samples to be culture positive, and the MB/BacT system increased the recovery up to 90 (66.7%) culture positives. On the other hand, PCR yielded 93 (68.9%) positive results, 20 (21.5%) of which were culture-negative sputum specimens. CONCLUSION: It is suggested that the Z-N direct microscopy on its own is the best method (with high specificity) for confirming the diagnosis of acid-fast bacilli. Although the PCR diagnosis of TB appears to be a rapid and sensitive method, the results should be interpreted with care in the clinical settings.