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Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)–bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non–small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs.Significance:This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy.

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In this study, we characterized nuclear morphology and function as cancer cells underwent recovery following chemotherapeutic treatment to identify the unique characteristics associated with treatment resistance and successful survival. Cells that survived following treatment and resisted therapy-induced cell death were predominantly mononucleated with increased nuclear/cellular size, enabled by continuous endocycling. We found that cells that survive after therapy release likely employ more efficient DNA damage repair and exhibit a distinct nucleolar phenotype - fewer but larger nucleoli – and increased rRNA levels. These data support a paradigm where soon after therapy release, the treated population mostly contains cells with a high level of widespread and catastrophic DNA damage that leads to apoptosis, while the minority of cells that have successful DDR are more likely to access a pro-survival state. These findings suggest that one way cancer cells can survive systemic therapy is to enter the polyaneuploid cancer cell (PACC) state, a recently-described mechanism of therapy resistance. Cancer cells in this state are physically enlarged, undergo whole-genome doubling resulting in polyaneuploid genomes, and are associated with worse prognosis in cancer patients. The PACC state is accessed when a cancer cell experiences external stress, such as genotoxic chemotherapy; after a period of recovery, cells exit the PACC state and resume proliferation to repopulate the tumor cell pool. Our findings demonstrate the fate of cancer cells following chemotherapy treatment and define key characteristics of the resistant PACC state. This work is essential for understanding and, ultimately, targeting, cancer resistance and recurrence.

13 páginas, 7 figuras, Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg., This study was supported by Taiwan Ministry of Science and Technology grants MOST 104-2320-B-002-044-MY3, MOST 106-2320-B-002-046-MY3, and MOST 108-2320-B-002-024-MY3, National Health Research Institutes grants NHRI-EX106-10401BI and NHRI-EX109-10725BI, National Taiwan University grants NTU107L890504 and NTU110L893503 to M.-S. Lee, and National Taiwan University Hospital grants 106-003451, 107-003849, 108-004269, and 109-004720 to C.-C. Ho. This work was also supported by MINECO grants BFU2016-80570-R and RTI2018-098084-B-I00 (AEI/FEDER, UE). The authors would like to thank the Laboratory Animal Core Facility at the College of Medicine, National Taiwan University for their services

Once prostate cancer has spread from its primary site, treatment is limited to systemically administered therapy. While tumors initially respond to these therapies, eventually, all therapies fail, and the tumor recurs. Ours and others' recent studies have shown the presence of a universal mechanism of resistance: accession of the polyaneuploid cancer cell (PACC) state. The PolyAneuploid Transition (PAT) is initiated when a cancer cell is subjected to external stress, including therapy (we have tested three classes of chemotherapy and radiation), and accesses an alternative non-mitotic cell cycle known as endocycling, resulting in a greater-than-G2 polyploidization of the aneuploid genome (polyaneuploidy). While in this pro-survival PACC state, we have shown that cells are resistant to all forms of cytotoxic therapy. Initial RNA sequencing of cells that have accessed the PACC state in response to multiple classes of chemotherapy, including docetaxel and cisplatin, demonstrate a remodeled transcriptional profile that is distinct from control cultures. We hypothesize that accession of the resistance-mediating PACC state requires transcriptional remodeling. Our data show that cells in the PACC state had increased overall transcriptional activity. The multi protein complex pTEFB (positive transcription elongation factor) is a critical driver of RNA Polymerase II function and is composed of multiple subunits, including CDK9, a cyclin regulatory subunit, and a transcription factor. CDK9 phosphorylation was significantly elevated during accession and persistence in the PACC state. Genetic inhibitionof CDK9 by siRNA led to decreased expression of cyclin A which plays a role in S phase, both in the mitoticcycle and in the PACC endocycle. Interestingly, despite increased CDK9 levels, known CDK9 pTEFb complex members cyclin T1 and BRD4 were both decreased in cells in the PACC state. We treated cancer cells induced to enter the PACC state upon exposure to cisplatin with pharmaceutical inhibitors of CDK9, Flavopiridol and Fadraciclib. Combination of CDK9 inhibition with chemotherapy showed a suppressive effect on the PACC population. Our findings therefore suggest that a non-canonical CDK9 complex could be critical role for the endocycling to access and maintain the PACC state and represents a therapeutic candidate to eliminate the resistance-mediating PACC state. Citation Format: Cheng-fan Lee, Michael Loycano, Luke LOFTUS, Laurie Kostecka, Melvin Li, Sarah Amend, Kenneth Pienta. A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5988.

Preferential wasting of the thenar muscles, the split-hand sign, may be used for early diagnosis of amyotrophic lateral sclerosis (ALS).Electronic databases were searched for studies assessing the split-hand index (SHI) and the compound muscle action potential (CMAP) amplitudes of abductor pollicis brevis (APB), first dorsal interosseous (FDI), and abductor digiti minimi (ADM). The SHI was obtained by multiplying CMAP amplitudes of APB and FDI and dividing the product by the CMAP amplitude of ADM. The Bayesian analysis was used for validation.In total, 17 studies and 1635 patients were included. Our meta-analysis revealed that ALS patients had significantly decreased SHI (standardized mean difference [SMD], -1.60, P 0.001), CMAP of the APB (SMD, -1.67, P 0.001), FDI (SMD, -1.12, P 0.001), and ADM (SMD, -1.09, P 0.001). The binormal receiver operating characteristic curve analysis showed a threshold of 7.4 for SHI, and cutoff values of 6.4 mV for APB and 8.4 mV for FDI, respectively. The Bayesian analysis validated decreased SHI in ALS patients (posterior mean difference of - 5.91).An SHI of 7.4 can be used facilitating earlier diagnosis of ALS.SHI can be used as a standard neurophysiological biomarker for early diagnosis.

Aberrant expression of transforming growth factor-β1 (TGF-β1) is associated with renal cell carcinoma (RCC) progression by inducing cancer metastasis. However, the downstream effector(s) in TGF-β signaling pathway is not fully characterized. In the present study, the elevation of secreted protein acidic and rich in cysteine (SPARC) as a TGF-β regulated gene in RCC was identified by applying differentially expressed gene analysis and microarray analysis, we further confirmed this result in several RCC cell lines. Clinically, the expression of these two genes is positively correlated in RCC patient specimens. Furthermore, elevated SPARC expression is found in all the subtypes of RCC and positively correlated with the RCC stage and grade. In contrast, SPARC expression is inversely correlated with overall and disease-free survival of patients with RCC, suggesting SPARC as a potent prognostic marker of RCC patient survival. Knocking down SPARC significantly inhibits RCC cell invasion and metastasis both in vitro and in vivo. Similarly, in vitro cell invasion can be diminished by using a specific monoclonal antibody. Mechanistically, SPARC activates protein kinase B (AKT) pathway leading to elevated expression of matrix metalloproteinase-2 that can facilitate RCC invasion. Altogether, our data support that SPARC is a critical role of TGF-β signaling network underlying RCC progression and a potential therapeutic target as well as a prognostic marker.

TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2’s cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.

Neuroendocrine prostate cancer (NEPC) is often diagnosed as a sub-type from the castration-resistant prostate cancer (CRPC) recurred from the second generation of anti-androgen treatment and is a rapidly progressive fatal disease. The molecular mechanisms underlying the trans-differentiation from CRPC to NEPC are not fully characterized, which hampers the development of effective targeted therapy.Bioinformatic analyses were conducted to determine the clinical correlation of sphingosine kinase 1 (SphK1) in CRPC progression. To investigate the transcriptional regulation SphK1 and neuroendocrine (NE) transcription factor genes, both chromosome immunoprecipitation and luciferase reporter gene assays were performed. To demonstrate the role of SphK1 in NEPC development, neurosphere assay was carried out along with several biomarkers determined by quantitative PCR and western blot. Furthermore, in vivo NEPC xenograft models and patient-derived xenograft (PDX) model were employed to determine the effect of SphK1 inhibitors and target validation.Significant prevalence of SphK1 in NEPC development is observed from clinical datasets. SphK1 is transcriptionally repressed by androgen receptor-RE1-silencing transcription factor (REST) complex. Furthermore, sphingosine 1-phosphate produced by SphK1 can modulate REST protein turnover via MAPK signaling pathway. Also, decreased REST protein levels enhance the expression of NE markers in CRPC, enabling the transition to NEPC. Finally, specific SphK1 inhibitors can effectively inhibit the growth of NEPC tumors and block the REST protein degradation in PDX.SphK1 plays a central role in NEPC development, which offers a new target for this lethal cancer using clinically approved SphK1 inhibitors.

Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-β action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-β1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-β/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.

Beyond its well-known canonical function as a tumor suppressor, p53 is also involved in numerous cellular processes through altered transcription under both normal and pathological conditions. The functional diversity of p53 outputs is complex and dependent on cell context. However, the underlying mechanisms responsible for this diversity remain largely unclear. The emerging evidence of p53 mutations involved in regulating endocytic trafficking and signaling, in tandem to promote malignancy (invasion, exosome biogenesis and immune evasion), sheds light on possible mechanisms behind the p53-driven complexity. The interrelated nature of endocytic trafficking and receptor signaling that form dynamic and adaptable feedback loops - either positive or negative - functions to modulate multiple cellular outputs. Biasing the tunable endocytic trafficking and receptor signaling network by mutant p53 expands the purview of p53, allowing its contribution to diverse and aggressive phenotypes. In this review, we explore recent studies in which the novel role of mutant p53 in altering endocytic trafficking to bias receptor signaling and drive transforming phenotypes is revealed. Understanding the complex crosstalk of mutant p53, endocytic trafficking and receptor signaling will allow the development of therapies to selectively target p53-altered endocytic processes.

Background: Aberrant expression of transforming growth factorβ1 (TGF-β1) is associated with renal cell carcinoma (RCC) progression by inducing cancer metastasis. However, the downstream effector(s) in TGF-β signaling pathway that is involved in RCC progression is not fully characterized. Methods: SPARC was identified as a potential downstream target gene in TGF-β-induced cell invasion by applying microarray analysis, qRT-PCR and Western blot. In vitro and in vivo assays were subsequently performed to validate the role of SPARC in RCC. SPARC expression profile in RCC tissues and its correlation with clinical data were also examined. Findings: We demonstrate that elevated SPARC is associated with metastatic RCC; SPARC gene expression can be induced by TGF-β1 via Snail. Clinically, the expression of these two genes is positively correlated in RCC patient specimens. Furthermore, elevated SPARC expression is found in all the subtypes of RCC and positively correlated with RCC stage and grade. In contrast, SPARC expression is inversely correlated with overall and disease-free survival of RCC patients, suggesting SPARC as a potent prognostic marker of RCC patient survival. Knocking down SPARC significantly inhibits RCC cell invasion and metastasis both in vitro and in vivo. Similarly, in vitro cell invasion can be diminished by using specific monoclonal antibody. Mechanistically, SPARC activate AKT pathway leading to elevated expression of matrix metalloproteinase-2 (MMP2) that can facilitate RCC invasion. Interpretation: Our data support that SPARC regulated by TGF-β is a key driver for RCC metastases, therefore, it becomes a potent therapeutic target. Funding Statement: This study was in part funded by a grant from China Scholarship Council (201508440279) and a grant from Health Commission of Guangdong Province (A2019513). This work was supported in part by the Harold C. Simmons Cancer Center through an NCI Cancer Center Support Grant, 1P30 CA142543 and The Department of Radiology. Declaration of Interests: All the authors do not have any conflict of interest. Ethics Approval Statement: The Institutional Review Board approved clinical specimen procurement protocol for this study and informed consent was obtained from patients. Animal work described in this manuscript has been approved and conducted under the oversight of the UTSW Institutional Animal Care and Use Committee.

Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.

A 15-year cohort study indicates that bacterial-induced prostatitis is correlated to prostate cancer (PCa) incidence. Several gram-negative bacteria have been identified in PCa patient specimens and inflammatory cytokines derived from bacterial infection have been involved in PCa tumorigenesis. However, the direct effect of lipopolysaccharide (LPS), a potent inflammatory substance from gram-negative bacteria, on PCa development is not well studied. In this study, we examine the effect of LPS on the growth (MTT assay) and invasion (Transwell assay) of several PCa cells lines including LNCaP, PC3, and DU145. Although LPS does not change the cell growth, our results reveal that LPS can enhance the PCa cell invasiveness and increase pericellular proteolytic activities. Using protease inhibitor screening and proteolytic analyses, we have identified that matriptase (MTX), a member of the membrane-anchored serine protease family, plays a key role in LPS-induced PCa cell invasiveness. The activated levels of MTX can be detected in the cells and the conditioned medium (CM) from LPS-treated cells. Indeed, the elevation of MTX expression is associated with high-grade PCa in archival specimens. Knowing LPS as a ligand for toll-like receptor 4 (TLR4), specific antagonists and shRNA knockdown methodology were used to delineate the mechanism by which TLR4/sphingosine kinase 1 (SphK1) signaling cascade plays a role in LPS-induced PCa cell invasion. The results show that SphK1 was important for MTX activation and PCa cell invasion. Moreover, we found that LPS could induce the activation of SphK1 based on an elevated phosphorylation level and SphK1 translocation to plasma membrane for the production of sphingosine 1-phosphate (S1P). S1P can subsequently induce MTX activation and PCa cell invasion through S1P receptor 4 (S1PR4) in a paracrine or autocrine manner. S1PR4 is further identified as an important receptor for S1P to mediate MAPK signaling. Furthermore, there is a significant correlation between the elevated expression of SphK1 and the Gleason scores of PCa specimens. The phenomenon was further corroborated by the fact that LPS could induce SphK1 activation in patient-derived explant model (PDeX). Taken together, we have unveiled a novel LPS-elicited pathway leading to PCa cell invasion. Our results provide not only the potential impact of bacterial infection on PCa progression but also several therapeutic target(s) against PCa metastasis. Citation Format: Cheng-Fan Lee, Benjamin Chen, Jer-Tsong Hsieh, Ming-Shyue Lee. Activation of sphingosine kinase 1 by lipopolysaccharide promotes prostate cancer cell invasion via matriptase [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A081.

Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.