Keratin 17 modulates the immune topography of pancreatic cancer.

BACKGROUND: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. METHODS: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. RESULTS: K17 expression had profound effects on the exclusion of intratumoral CD8+ T cells and was also associated with decreased numbers of peritumoral CD8+ T cells, CD16+ macrophages, and CD163+ macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8+ T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. CONCLUSIONS: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.

Keratin 17 is a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma.

OBJECTIVES: To determine the role of keratin 17 (K17) as a predictive biomarker for response to chemotherapy by defining thresholds of K17 expression based on immunohistochemical tests that could be used to optimize therapeutic intervention for patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: We profiled K17 expression, a hallmark of the basal molecular subtype of PDAC, by immunohistochemistry in 2 cohorts of formalin-fixed, paraffin-embedded PDACs (n = 305). We determined a K17 threshold of expression to optimize prognostic stratification according to the lowest Akaike information criterion and explored the potential relationship between K17 and chemoresistance by multivariate predictive analyses. RESULTS: Patients with advanced-stage, low K17 PDACs treated using 5-fluorouracil (5-FU)-based chemotherapeutic regimens had 3-fold longer survival than corresponding cases treated with gemcitabine-based chemotherapy. By contrast, PDACs with high K17 did not respond to either regimen. The predictive value of K17 was independent of tumor mutation status and other clinicopathologic variables. CONCLUSIONS: The detection of K17 in 10% or greater of PDAC cells identified patients with shortest survival. Among patients with low K17 PDACs, 5-FU-based treatment was more likely than gemcitabine-based therapies to extend survival.

Keratin 17 modulates the immune topography of pancreatic cancer.

Background: The immune microenvironment impacts tumor growth, invasion, metastasis, and patient survival and may provide opportunities for therapeutic intervention in pancreatic ductal adenocarcinoma (PDAC). Although never studied as a potential modulator of the immune response in most cancers, Keratin 17 (K17), a biomarker of the most aggressive (basal) molecular subtype of PDAC, is intimately involved in the histogenesis of the immune response in psoriasis, basal cell carcinoma, and cervical squamous cell carcinoma. Thus, we hypothesized that K17 expression could also impact the immune cell response in PDAC, and that uncovering this relationship could provide insight to guide the development of immunotherapeutic opportunities to extend patient survival. Methods: Multiplex immunohistochemistry (mIHC) and automated image analysis based on novel computational imaging technology were used to decipher the abundance and spatial distribution of T cells, macrophages, and tumor cells, relative to K17 expression in 235 PDACs. Results: K17 expression had profound effects on the exclusion of intratumoral CD8 + T cells and was also associated with decreased numbers of peritumoral CD8 + T cells, CD16 + macrophages, and CD163 + macrophages (p < 0.0001). The differences in the intratumor and peritumoral CD8 + T cell abundance were not impacted by neoadjuvant therapy, tumor stage, grade, lymph node status, histologic subtype, nor KRAS, p53, SMAD4, or CDKN2A mutations. Conclusions: Thus, K17 expression correlates with major differences in the immune microenvironment that are independent of any tested clinicopathologic or tumor intrinsic variables, suggesting that targeting K17-mediated immune effects on the immune system could restore the innate immunologic response to PDAC and might provide novel opportunities to restore immunotherapeutic approaches for this most deadly form of cancer.

Dissecting the Oncogenic Roles of Keratin 17 in the Hallmarks of Cancer.

There is an unmet need to identify and validate tumor-specific therapeutic targets to enable more effective treatments for cancer. Heterogeneity in patient clinical characteristics as well as biological and genetic features of tumors present major challenges for the optimization of therapeutic interventions, including the development of novel and more effective precision medicine. The expression of keratin 17 (K17) is a hallmark of the most aggressive forms of cancer across a wide range of anatomical sites and histological types. K17 correlates with shorter patient survival, predicts resistance to specific chemotherapeutic agents, and harbors functional domains that suggest it could be therapeutically targeted. Here, we explore the role of K17 in the hallmarks of cancer and summarize evidence to date for K17-mediated mechanisms involved in each hallmark, elucidating functional roles that warrant further investigation to guide the development of novel therapeutic strategies.

Keratin 17 Is a Novel Cytologic Biomarker for Urothelial Carcinoma Diagnosis.

OBJECTIVES: The microscopic features of urine cytology specimens are subjective and may not reliably distinguish between benign urothelial cells and low-grade urothelial carcinoma (UC). Prior studies demonstrated that keratin 17 (K17) detection in biopsies is highly sensitive for UC. The current study aimed to define K17 diagnostic test performance for initial screening and detect recurrent UC in urine specimens. METHODS: K17 was detected by immunocytochemistry (ICC) in consecutively collected urine specimens (2018-2019). A qualitative score for the K17 test was determined in 81 samples (discovery cohort) and validated in 98 samples (validation cohort). K17 sensitivity and specificity were analyzed in both cohorts across all grades of UC. RESULTS: Based on the discovery cohort, the presence of 5 or more K17 immunoreactive urothelial cells (area under the curve = 0.90; P < .001) was the optimal threshold to define a K17-positive test. The sensitivity of the K17 ICC test for biopsy-confirmed UC was 35 of 36 (97%) and 18 of 21 (86%) in the discovery and validation cohorts, respectively. K17 was positive in 16 of 19 (84%) specimens with biopsy-confirmed low-grade UC and in 34 of 34 (100%) of specimens with high-grade UC. CONCLUSIONS: K17 ICC is a highly sensitive diagnostic test for initial screening and detection of recurrence across all grades of UC.

Keratin 17 testing in pancreatic cancer needle aspiration biopsies predicts survival.

BACKGROUND: Although pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers, differences in survival exist between patients with clinically identical characteristics. The authors previously demonstrated that keratin 17 (K17) expression in PDAC, measured by RNA sequencing or immunohistochemistry (IHC), is an independent negative prognostic biomarker. Only 20% of cases are candidates for surgical resection, but most patients are diagnosed by needle aspiration biopsy (NAB). The aims of this study were to determine whether there was a correlation in K17 scores detected in matched NABs and surgical resection tissue sections and whether K17 IHC in NAB cell block specimens could be used as a negative prognostic biomarker in PDAC. METHODS: K17 IHC was performed for a cohort of 70 patients who had matched NAB cell block and surgical resection samples to analyze the correlation of K17 expression levels. K17 IHC was also performed in cell blocks from discovery and validation cohorts. Kaplan-Meier and Cox proportional hazards regression models were analyzed to determine survival differences in cases with different levels of K17 IHC expression. RESULTS: K17 IHC expression correlated in matched NABs and resection tissues. NAB samples were classified as high for K17 when ≥80% of tumor cells showed strong (2+) staining. High-K17 cases, including stage-matched cases, had shorter survival. CONCLUSIONS: K17 has been identified as a robust and independent prognostic biomarker that stratifies clinical outcomes for cases that are diagnosed by NAB. Testing for K17 also has the potential to inform clinical decisions for optimization of chemotherapeutic interventions.

Pancreatic cancer driver mutations are targetable through distant alternative RNA splicing dependencies.

Pancreatic ductal adenocarcinoma (PDAC), the most common histological subtype of pancreatic cancer, has one of the highest case fatality rates of all known solid malignancies. Over the past decade, several landmark studies have established mutations in KRAS and TP53 as the predominant drivers of PDAC pathogenesis and therapeutic resistance, though treatment options for PDACs and other tumors with these mutations remain extremely limited. Hampered by late tumor discovery and diagnosis, clinicians are often faced with using aggressive and non-specific chemotherapies to treat advanced disease. Clinically meaningful responses to targeted therapy are often limited to the minority of patients with susceptible PDACs, and immunotherapies have routinely encountered roadblocks in effective activation of tumor-infiltrating immune cells. Alternative RNA splicing (ARS) has recently gained traction in the PDAC literature as a field from which we may better understand and treat complex mechanisms of PDAC initiation, progression, and therapeutic resistance. Here, we review PDAC pathogenesis as it relates to fundamental ARS biology, with an extension to implications for PDAC patient clinical management.

The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma.

Pancreatic adenosquamous carcinoma (PASC) is an aggressive cancer whose mutational origins are poorly understood. An early study reported high-frequency somatic mutations affecting UPF1, a nonsense-mediated mRNA decay (NMD) factor, in PASC, but subsequent studies did not observe these lesions. The corresponding controversy about whether UPF1 mutations are important contributors to PASC has been exacerbated by a paucity of functional studies. Here, we modeled two UPF1 mutations in human and mouse cells to find no significant effects on pancreatic cancer growth, acquisition of adenosquamous features, UPF1 splicing, UPF1 protein, or NMD efficiency. We subsequently discovered that 45% of UPF1 mutations reportedly present in PASCs are identical to standing genetic variants in the human population, suggesting that they may be non-pathogenic inherited variants rather than pathogenic mutations. Our data suggest that UPF1 is not a common functional driver of PASC and motivate further attempts to understand the genetic origins of these malignancies.

Cancer is a group of complex diseases in which cells grow uncontrollably and spread into surrounding tissues and other parts of the body. All types of cancers develop from changes ­ or mutations ­ in the genes that affect the pathways involved in controlling the growth of cells. Different cancers possess unique sets of mutations that affect specific genes, and often, it is difficult to determine which of them play the most important role in a particular type of cancer. For example, pancreatic adenosquamous carcinoma, a rare and aggressive form of pancreatic cancer, is a devastating disease with a poor chance of survival ­ patients rarely live longer than one year after diagnosis. While the cells of this particular cancer display distinct features that separate them from other forms of pancreatic cancer, the genetic causes of these features are unclear. Using new technologies, some researchers have reported mutations in a 'quality control' gene called 'UPF1', which is responsible for destroying faulty forms of genetic material. However, subsequent studies did not find such mutations. To clarify the role of UPF1 in pancreatic adenosquamous carcinoma, Polaski et al. used mouse and human cancer cells with UPF1 mutations and monitored their effects on tumour growth and the development of features unique to this disease. Polaski et al. first injected mice with mouse pancreatic cancer cells containing mutations in UPF1 (mutated cells) and cancer cells without. Both groups of mice developed pancreatic tumours but there was no difference in tumour growth between the mutated and non-mutated cells, and neither cell type displayed distinct features. The researchers then generated human mutated cells, which were also found to lack any specific characteristics. Further analysis showed that the mutations did not stop UPF1 from working, in fact, over 40% of these mutations occurred naturally in humans without causing cancer. This suggests that UPF1 does not seem to be involved in pancreatic adenosquamous carcinoma. Further investigation is needed to illuminate key genetic players in the development of this type of cancer, which will be vital for improving treatments and outcomes for patients suffering from this disease.

Keratin 17 Expression Predicts Poor Clinical Outcome in Patients With Advanced Esophageal Squamous Cell Carcinoma.

The major roles of keratin 17 (K17) as a prognostic biomarker have been highlighted in a range of human malignancies. However, its relevance to esophageal squamous cell carcinoma (ESCC) remains unexplored. In this study, the relationship between K17 expression and clinicopathologic parameters and survival were determined by RNA sequencing (RNA-Seq) in 90 ESCCs and by immunohistochemistry (IHC) in 68 ESCCs. K17 expression was significantly higher in ESCC than in paired normal tissues at both the messenger RNA and protein levels. K17 messenger RNA and staining by IHC were significantly correlated with aggressive characteristics, including advanced clinical stage, invasion depth, and lymph node metastases; and were predictive of poor prognosis in advanced disease patients. Furthermore, K17 expression was detected by IHC in high-grade premalignant lesions of the esophageal mucosa, suggesting that K17 could also be a biomarker of dysplasia of the esophageal mucosa. Overall, this study established that K17 is a negative prognostic biomarker for the most common subtype of esophageal cancer.

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

An unbiased high-throughput drug screen reveals a potential therapeutic vulnerability in the most lethal molecular subtype of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related deaths in the United States by 2020, due in part to innate resistance to widely used chemotherapeutic agents and limited knowledge about key molecular factors that drive tumor aggression. We previously reported a novel negative prognostic biomarker, keratin 17 (K17), whose overexpression in cancer results in shortened patient survival. In this study, we aimed to determine the predictive value of K17 and explore the therapeutic vulnerability in K17-expressing PDAC, using an unbiased high-throughput drug screen. Patient-derived data analysis showed that K17 expression correlates with resistance to gemcitabine (Gem). In multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells, and orthotopic xenografts, we determined that the expression of K17 results in a more than twofold increase in resistance to Gem and 5-fluorouracil, key components of current standard-of-care chemotherapeutic regimens. Furthermore, through an unbiased drug screen, we discovered that podophyllotoxin (PPT), a microtubule inhibitor, showed significantly higher sensitivity in K17-positive compared to K17-negative PDAC cell lines and animal models. In the clinic, another microtubule inhibitor, paclitaxel (PTX), is used in combination with Gem as a first-line chemotherapeutic regimen for PDAC. Surprisingly, we found that when combined with Gem, PPT, but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells. Importantly, in preclinical models, PPT in combination with Gem effectively decreased tumor growth and enhanced the survival of mice bearing K17-expressing tumors. This provides evidence that PPT and its derivatives could potentially be combined with Gem to enhance treatment efficacy for the ~ 50% of PDACs that express high levels of K17. In summary, we reported that K17 is a novel target for developing a biomarker-based personalized treatment for PDAC.

Keratin 17 is a negative prognostic biomarker in high-grade endometrial carcinomas.

Keratin 17 (K17) has been established as a negative prognostic biomarker in cervical and ovarian cancers but has not previously been evaluated as a prognostic biomarker in endometrial adenocarcinoma. The association of K17 with decreased patient survival may be explained in part by the discovery that K17 drives tumor aggression by serving as a nuclear shuttle of p27, leading to cell cycle progression and tumor growth. The current study tests the hypothesis that K17 mRNA and protein levels correlate with decreased survival of patients with high-grade endometrial cancer. Gene expression data (mRNA) from The Cancer Genome Atlas were analyzed for 271 high-grade endometrial carcinomas and K17 immunohistochemistry (IHC) was performed on a separate cohort of 119 high-grade endometrial cancer cases from two academic medical centers. Survival analyses were determined by Cox proportional hazards regression. High K17 mRNA and IHC correlated with decreased overall survival (HR: 1.8, P = .0101, HR: 1.8, P = .0488, respectively). K17 was positive in malignant glandular cells of the endometrium but not in other tissues, including endometrial stroma, myometrium and uterine sarcoma. These results support the conclusion that K17 is a negative prognostic biomarker in high-grade endometrial carcinoma and that K17 IHC test results could be used to inform decisions related to therapeutic intervention.

Keratin 17 identifies the most lethal molecular subtype of pancreatic cancer.

Although the overall five-year survival of patients with pancreatic ductal adenocarcinoma (PDAC) is dismal, there are survival differences between cases with clinically and pathologically indistinguishable characteristics, suggesting that there are uncharacterized properties that drive tumor progression. Recent mRNA sequencing studies reported gene-expression signatures that define PDAC molecular subtypes that correlate with differences in survival. We previously identified Keratin 17 (K17) as a negative prognostic biomarker in other cancer types. Here, we set out to determine if K17 is as accurate as molecular subtyping of PDAC to identify patients with the shortest survival. K17 mRNA was analyzed in two independent PDAC cohorts for discovery (n = 124) and validation (n = 145). Immunohistochemical localization and scoring of K17 immunohistochemistry (IHC) was performed in a third independent cohort (n = 74). Kaplan-Meier and Cox proportional-hazard regression models were analyzed to determine cancer specific survival differences in low vs. high mRNA K17 expressing cases. We established that K17 expression in PDACs defines the most aggressive form of the disease. By using Cox proportional hazard ratio, we found that increased expression of K17 at the IHC level is also associated with decreased survival of PDAC patients. Additionally, within PDACs of advanced stage and negative surgical margins, K17 at both mRNA and IHC level is sufficient to identify the subgroup with the shortest survival. These results identify K17 as a novel negative prognostic biomarker that could inform patient management decisions.

Keratin 17 is a sensitive and specific biomarker of urothelial neoplasia.

There is a clinical need to identify novel biomarkers to improve diagnostic accuracy for the detection of urothelial tumors. The current study aimed to evaluate keratin 17 (K17), an oncoprotein that drives cell cycle progression in cancers of multiple anatomic sites, as a diagnostic biomarker of urothelial neoplasia in bladder biopsies and in urine cytology specimens. We evaluated K17 expression by immunohistochemistry in formalin-fixed, paraffin embedded tissue specimens of non-papillary invasive urothelial carcinoma (UC) (classical histological cases), high grade papillary UC (PUC-LG), low grade papillary UC (PUC-HG), papillary urothelial neoplasia of low malignant potential (PUNLMP), and normal bladder mucosa. A threshold was established to dichotomize K17 status in tissue specimens as positive vs. negative, based on the proportion of cells that showed strong staining. In addition, K17 immunocytochemistry was performed on urine cytology slides, scoring positive test results based on the detection of K17 in any urothelial cells. Mann-Whitney and receiver operating characteristic analyses were used to compare K17 expression between histologic diagnostic categories. The median proportion of K17 positive tumor cells was 70% (range 20-90%) in PUNLMP, 30% (range 5-100%) in PUC-LG, 20% (range 1-100%), in PUC-HG, 35% (range 5-100%) in UC but staining was rarely detected (range 0-10%) in normal urothelial mucosa. Defining cases in which K17 was detected in ≥10% of cells were considered positive, the sensitivity of K17 in biopsies was 89% (95% CI: 80-96%) and the specificity was 88% (95% CI: 70-95%) to distinguish malignant lesions (PUC-LG, PUC-HG, and UC) from normal urothelial mucosa. Furthermore, K17 immunocytochemistry had a sensitivity of 100% and a specificity of 96% for urothelial carcinoma in 112 selected urine specimens. Thus, K17 is a sensitive and specific biomarker of urothelial neoplasia in tissue specimens and should be further explored as a novel biomarker for the cytologic diagnosis of urine specimens.

Elevated expression of keratin 17 in oropharyngeal squamous cell carcinoma is associated with decreased survival.

BACKGROUND: Overexpression of keratin 17 (K17) is highly associated with poor prognosis in squamous cell carcinoma (SCC) of the cervix. This study was performed to (1) determine whether K17 may be a prognostic biomarker in head and neck squamous cell carcinoma (HNSCC) and (2) to establish if K17 expression is associated with human papillomavirus (HPV) status. METHODS: Immunohistochemical staining was performed for K17 of oral, oropharyngeal, and laryngeal SCCs, and normal oropharyngeal mucosa. The HPV status was determined using polymerase chain reaction (PCR). RESULTS: Elevated K17 expression was significantly associated with an overall decreased patient survival (P = .02) and, more specifically, in patients with oropharyngeal SCC (P = .01). When controlling for HPV status and tumor location K17 was still a significant predictor of survival (P = .01). CONCLUSION: Therefore, K17 is a novel prognostic biomarker of poor survival for patients with HNSCCs, controlling for anatomic site and HPV status.

Keratin 17 Is a Prognostic Biomarker in Endocervical Glandular Neoplasia.

OBJECTIVES: Previous work in our laboratory identified keratin 17 (K17) as a specific and sensitive biomarker for high-grade squamous intraepithelial lesions and cervical squamous cell carcinoma (SCC). K17, however, has not been previously evaluated in endocervical glandular neoplasia. Based on the similar pathogenesis of squamous and glandular lesions of the cervix, we hypothesized that K17 overexpression could also be a diagnostic and/or prognostic biomarker for endocervical neoplasia. METHODS: Cases of endocervical adenocarcinoma (n = 90), adenocarcinoma in situ (AIS) (n = 32), benign glandular lesions (n = 36), and normal endocervical mucosa (n = 5) were selected from Stony Brook Medicine and the University of Massachusetts from 2002 to 2013. Immunohistochemical staining for K17 was performed by an indirect immunoperoxidase method and was scored based on the proportion of cells that showed strong (2+) staining. RESULTS: K17 was highly expressed in 21 (65.6%) of 32 AIS and in 75 (83.0%) of 90 adenocarcinoma cases. In adenocarcinomas, K17 staining was detected in a mean of 33.9% of malignant cells. Staining tended to be strongest at the periphery of pseudoglandular groups and at the invasive front of tumors. K17 was not detected in the epithelial cells of benign glandular lesions, but groups of cuboidal cells, residing beneath the epithelial layer of benign glands, were frequently positive for K17, especially in cases of microglandular hyperplasia. High levels of K17 expression were significantly associated with decreased patient survival. CONCLUSIONS: K17 is highly expressed in most cases of both invasive adenocarcinoma and in AIS and is a powerful, negative prognostic marker for patient survival.

Keratin 17 is overexpressed and predicts poor survival in estrogen receptor-negative/human epidermal growth factor receptor-2-negative breast cancer.

Clinicopathological features of breast cancer have limited accuracy to predict survival. By immunohistochemistry (IHC), keratin 17 (K17) expression has been correlated with triple-negative status (estrogen receptor [ER]/progesterone receptor/human epidermal growth factor receptor-2 [HER2] negative) and decreased survival, but K17 messenger RNA (mRNA) expression has not been evaluated in breast cancer. K17 is a potential prognostic cancer biomarker, targeting p27, and driving cell cycle progression. This study compared K17 protein and mRNA expression to ER/progesterone receptor/HER2 receptor status and event-free survival. K17 IHC was performed on 164 invasive breast cancers and K17 mRNA was evaluated in 1097 breast cancers. The mRNA status of other keratins (16/14/9) was evaluated in 113 ER-/HER2- ductal carcinomas. IHC demonstrated intense cytoplasmic and membranous K17 localization in myoepithelial cells of benign ducts and lobules and tumor cells of ductal carcinoma in situ. In ductal carcinomas, K17 protein was detected in most triple-negative tumors (28/34, 82%), some non-triple-negative tumors (52/112, 46%), but never in lobular carcinomas (0/15). In ductal carcinomas, high K17 mRNA was associated with reduced 5-year event-free survival in advanced tumor stage (n = 149, hazard ratio [HR] = 3.68, P = .018), and large (n = 73, HR = 3.95, P = .047), triple-negative (n = 103, HR = 2.73, P = .073), and ER-/HER2- (n = 113, HR = 2.99, P = .049) tumors. There were significant correlations among keratins 17, 16, 14, and 9 mRNA levels suggesting these keratins (all encoded on chromosome 17) could be coordinately expressed in breast cancer. Thus, K17 is expressed in a subset of triple-negative breast cancers, and is a marker of poor prognosis in patients with advanced stage and ER-/HER2- breast cancer.

Laminin promotes metalloproteinase-mediated dystroglycan processing to regulate oligodendrocyte progenitor cell proliferation.

The cell surface receptor dystroglycan mediates interactions between oligodendroglia and laminin-211, an extracellular matrix protein that regulates timely oligodendroglial development. However, dystroglycan's precise role in oligodendroglial development and the potential mechanisms to regulate laminin-dystroglycan interactions remain unknown. Here we report that oligodendroglial dystroglycan is cleaved by metalloproteinases, thereby uncoupling oligodendroglia from laminin binding. Dystroglycan cleavage is selectively stimulated by oligodendrocyte progenitor cell attachment to laminin-211, but not laminin-111 or poly-D-lysine. In addition, dystroglycan cleavage occurs most prominently in oligodendrocyte progenitor cells, with limited dystroglycan cleavage observed in differentiating oligodendrocytes. When dystroglycan cleavage is blocked by metalloproteinase inhibitors, oligodendrocyte progenitor cell proliferation is substantially decreased. Conversely, expression of the intracellular portion of cleaved dystroglycan results in increased oligodendrocyte progenitor cell proliferation, suggesting that endogenous dystroglycan cleavage may promote oligodendrocyte progenitor cell cycle progression. Intriguingly, while matrix metalloproteinase-2 and/or -9 have been reported to be responsible for dystroglycan cleavage, we find that these two metalloproteinases are neither necessary nor sufficient for cleavage of oligodendroglial dystroglycan. In summary, laminin-211 stimulates metalloproteinase-mediated dystroglycan cleavage in oligodendrocyte progenitor cells (but not in differentiated oligodendrocytes), which in turn promotes oligodendrocyte progenitor cell proliferation. This novel regulation of oligodendroglial laminin-dystroglycan interactions may have important consequences for oligodendroglial differentiation, both during development and during disease when metalloproteinase levels become elevated.

Keratin-17 Promotes p27KIP1 Nuclear Export and Degradation and Offers Potential Prognostic Utility.

Keratins that are overexpressed selectively in human carcinomas may offer diagnostic and prognostic utility. In this study, we show that high expression of keratin-17 (K17) predicts poor outcome in patients with cervical cancer, at early or late stages of disease, surpassing in accuracy either tumor staging or loss of p27(KIP1) as a negative prognostic marker in this setting. We investigated the mechanistic basis for the biologic impact of K17 through loss- and gain-of-function experiments in human cervix, breast, and pancreatic cancer cells. Specifically, we determined that K17 functions as an oncoprotein by regulating the subcellular localization and degradation of p27(KIP1). We found that K17 was released from intermediate filaments and translocated into the nucleus via a nuclear localization signal (NLS), specific among keratins, where it bound p27(KIP1) during G1 phase of the cell cycle. p27(KIP1) lacks a nuclear export signal (NES) and requires an adaptor for CRM1 binding for nuclear export. In K17, we defined and validated a leucine-rich NES that mediated CRM1 binding for export. Cervical cancer cells expressing K17 mutations in its NLS or NES signals exhibited an increase in levels of nuclear p27(KIP1), whereas cells expressing wild-type K17 exhibited a depletion in total endogenous p27(KIP1). In clinical specimens of cervical cancer, we confirmed that the expressions of K17 and p27(KIP1) were inversely correlated, both across tumors and within individual tumors. Overall, our findings establish that K17 functions specially among keratins as an oncoprotein by controlling the ability of p27(KIP1) to influence cervical cancer pathogenesis.

Assessment of genomic damage and repair on human lymphocytes by paint thinner in vitro.

Paint thinners are organic-solvent complex mixtures frequently used by car painters around the world in industries and shops. Some studies have revealed the oxidative effect induced by thinner inhalation; however, its genotoxic effect is poorly studied. The aim of this study was to assess the cytotoxicity, genomic damage and DNA repair in vitro induced by commercial paint thinner 0.14 in human lymphocytes. Cytotoxicity was determined by cell-viability analysis with trypan blue after 4 h treatment with different thinner concentrations (0.025 to 1.2 µL/mL). Genomic damage was evaluated by means of the alkaline single-cell gel electrophoresis (SCGE; pH > 13) in treated cultures after 1 h with three low-cytotoxic thinner concentrations (0.05, 0.1 and 0.2 µL/mL). In order to evaluate the genomic DNA repair, one set of SCGE slides was prepared immediately after treatment, and another one was prepared after 4 h of liquid-holding recovery. A significant level of cytotoxicity was observed over the entire concentration range of paint thinner in lymphocytes (F = 175.98; p ≤ 0.001). In the SCGE % tail DNA assessment, a significant increase of lymphocyte genomic damage was evidenced (F = 72.32; p < 0.001). In addition, we found a significant decrease in the % tail DNA in thinner-treated cells after liquid-holding recovery period (all p < 0.05), demonstrating that DNA primary lesions induced by low-cytotoxic thinner concentrations are efficiently repaired. In conclusion, thinner components induce cytotoxicity and genomic damage in human lymphocytes under the study conditions, possibly by oxidative and alkylative DNA damage.