Elise Nilsson

Isolation and characterization of progenitor-like cells from human renal proximal tubules.

The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowmans capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.

Suppression of renal cell carcinoma growth by inhibition of Notch signaling in vitro and in vivo

Loss of the tumor suppressor gene von Hippel-Lindau (VHL) plays a key role in the oncogenesis of clear cell renal cell carcinoma (CCRCC). The loss leads to stabilization of the HIF transcription complex, which induces angiogenic and mitogenic pathways essential for tumor formation. Nonetheless, additional oncogenic events have been postulated to be required for the formation of CCRCC tumors. Here, we show that the Notch signaling cascade is constitutively active in human CCRCC cell lines independently of the VHL/HIF pathway. Blocking Notch signaling resulted in attenuation of proliferation and restrained anchorage-independent growth of CCRCC cell lines. Using siRNA targeting the different Notch receptors established that the growth-promoting effects of the Notch signaling pathway were attributable to Notch-1 and that Notch-1 knockdown was accompanied by elevated levels of the negative cell-cycle regulators p21 Cip1 and/or p27 Kip1. Treatment of nude mice with an inhibitor of Notch signaling potently inhibited growth of xenotransplanted CCRCC cells. Moreover, Notch-1 and the Notch ligand Jagged-1 were expressed at significantly higher levels in CCRCC tumors than in normal human renal tissue, and the growth of primary CCRCC cells was attenuated upon inhibition of Notch signaling. These findings indicate that the Notch cascade may represent a novel and therapeutically accessible pathway in CCRCC.

Multiple Cellular Mechanisms Related to Cyclin A1 in Prostate Cancer Invasion and Metastasis

BACKGROUND Cyclin A1 is a cell cycle regulator that has been implicated in the progression of prostate cancer. Its role in invasion and metastasis of this disease has not been characterized. METHODS Immunohistochemistry and cDNA microarray analyses were used to assess protein and mRNA expression of cyclin A1 and proteins with roles in metastasis, including vascular endothelial growth factor (VEGF), metalloproteinase 2 (MMP2), and MMP9, in human prostate cancer. Transient transfection and infection with viral vectors expressing cyclin A1 and short hairpin RNA (shRNA) targeting cyclin A1 were used to study the effects of altered cyclin A1 expression in PC3 prostate cancer cells. The BrdU assay, annexin V staining, and invasion chambers were used to examine cyclin A1 effects on proliferation, apoptosis, and invasion, respectively. The role of cyclin A1 and androgen receptor (AR) in transcription of VEGF and MMP2 was assessed by promoter mutation and chromatin immunoprecipitation. The effect of cyclin A1 expression on tumor growth and metastasis was analyzed in a mouse model of metastasis. All statistical tests were two-sided. RESULTS Cyclin A1 protein and mRNA expression were statistically significantly higher in prostate cancers than in adjacent benign tissues. A statistically significant correlation between expression of cyclin A1 and of MMP2, MMP9, and VEGF was observed in prostate tumors from 482 patients (P values from Spearman rank correlation tests < .001). PC3 cells that overexpressed cyclin A1 showed increased invasiveness, and inhibition of cyclin A1 expression via shRNA expression reduced invasiveness of these cells. Eight of 10 mice (80%) bearing PC3 cells overexpressing cyclin A1 had infiltration of tumor cells in lymph node, liver, and lung, but all 10 mice bearing tumors expressing control vector were free of liver and lung metastases and only one mouse from this group had lymph node metastasis (P values from Fisher exact tests < .001). Cyclin A1, in concert with AR, bound to and increased expression from the VEGF and MMP2 promoters. CONCLUSIONS Cyclin A1 contributes to prostate cancer invasion by modulating the expression of MMPs and VEGF and by interacting with AR.

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia‐inducible factor 1 (HIF‐1). HIF‐1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF‐1 and mediates the hypoxic repression of a set of nuclear‐encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear‐encoded mitochondrial genes where it directly antagonizes c‐Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short‐hairpin RNA (shRNA)‐expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.

ERK1/2 inhibition increases antiestrogen treatment efficacy by interfering with hypoxia-induced downregulation of ERalpha: a combination therapy potentially targeting hypoxic and dormant tumor cells.

Tumor hypoxia is associated with cancer invasiveness, metastasis and treatment failure. Recent data suggest that the major target for endocrine treatment in breast cancer, ERα, is downregulated during hypoxia, but the mechanism behind this remains unknown. MAPK signaling as well as ERα regulation has earlier been independently linked to hypoxia and we now demonstrate HIF-1α and ERK1/2-activation in vivo towards the necrotic zone in DCIS of the breast, parallel with ERα downregulation. Hypoxia further caused transcriptional downregulation of ERα via activation of ERK1/2 in cell lines and, importantly, MEK1/2 inhibitors (U0126 or PD184352) or ERK1/2 suppression by siRNA partially restored the ERα expression. U0126 combined with tamoxifen accordingly produced an increased efficacy of the anti-estrogens during hypoxia. Based on these findings, we suggest a promising novel therapy for ERα-positive breast cancer where a combination of endocrine treatment and ERK1/2 inhibitors may increase treatment response by improved targeting of dormant hypoxic tumor cells.

Increased androgen receptor expression in serous carcinoma of the ovary is associated with an improved survival.

BackgroundAltered androgen hormone homeostasis and androgen receptor (AR) activity have been implicated in ovarian carcinogenesis but the relationship between AR expression in ovarian cancer and clinical outcome remains unclear.MethodsIn this study, the prognostic impact of AR expression was investigated using immunohistochemistry in tissue microarrays from 154 incident cases of epithelial ovarian cancer (EOC) in the prospective, population-based cohorts Malmö Diet and Cancer Study and Malmö Preventive Project. A subset of corresponding fallopian tubes (n = 36) with no histopathological evidence of disease was also analysed.ResultsWhile abundantly expressed in the majority of fallopian tubes with more than 75% positive nuclei in 16/36 (44%) cases, AR was absent in 108/154 (70%) of EOC cases. AR expression was not related to prognosis in the entire cohort, but in the serous subtype (n = 90), AR positivity (> 10% positive nuclei) was associated with a prolonged disease specific survival in univariate (HR= 0.49; 95% CI 0.25-0.96; p= 0.038) and multivariate (HR= 0.46; 95% CI 0.22-0.97; p= 0.042) analysis, adjusted for age, grade and clinical stage.ConclusionsAR expression is considerably reduced in EOC as compared to fallopian tubes, and in EOC of the serous subtype, high AR expression is a favourable prognostic factor. These results indicate that assessment of AR expression might be of value for treatment stratification of EOC patients with serous ovarian carcinoma.

Increased serum levels of tumour-associated trypsin inhibitor independently predict a poor prognosis in colorectal cancer patients

BackgroundThere is an insufficient number of reliable prognostic and response predictive biomarkers in colorectal cancer (CRC) management. In a previous study, we found that high tumour tissue expression of tumour-associated trypsin inhibitor (TATI) correlated with liver metastasis and an impaired prognosis in CRC. The aim of this study was to investigate the prognostic validity of serum TATI (s-TATI) in CRC. We further assessed the prognostic value of carcino-embryonic antigen in serum (s-CEA) and the interrelationship between s-TATI and TATI in tissue (t-TATI).MethodsUsing an immunofluorometric assay, s-TATI levels were analysed in 334 preoperatively collected serum samples from patients with CRC. Spearmans Rho and Chi-square test were used for analysis of correlations between s-TATI and clinicopathological parameters, s-CEA and t-TATI. Kaplan-Meier analysis and Cox uni- and multivariate regression analysis were used to estimate disease free survival (DFS) and overall survival (OS) according to quartiles of s-TATI and cut-offs derived from ROC-analysis of s-TATI and s-CEA.ResultsIncreased levels of s-TATI were associated with a reduced DFS (HR = 2.00; 95% CI 1.40-2.84, P < 0.001) and OS (HR = 2.40; 95% CI 1.74-3.33, P < 0.001). (HR = 2.89; 95% CI 1.96-4.25). This association remained significant in multivariate analysis. The association for OS remained significant in multivariate analysis (HR = 1.51; 95% CI 1.03-2.22, P = 0.034 for DFS and HR = 1.78; 95% CI 1.25-2.53, P = 0.001 for OS). There was no significant association between s-TATI and t-TATI. The prognostic value of s-CEA was also evident, but somewhat weaker than for s-TATI.ConclusionsHigh preoperative s-TATI levels predict a poor prognosis in patients with CRC, and the prognostic value is independent of established prognostic parameters and t-TATI expression. These data suggest that s-TATI might be a useful marker for prognostic stratification in CRC.

The Bcl-xL inhibitor of apoptosis is preferentially expressed in cutaneous squamous cell carcinoma compared with that in keratoacanthoma.

Keratoacanthoma (KA) is difficult to histologically distinguish from squamous cell carcinoma (SCC). Therefore, although KA is a benign self‐resolving skin lesion, KA is commonly treated as SCC. Biomarkers to distinguish KA and SCC would thus be desirable. In search for specific markers, paraffin‐embedded tissue samples from 25 SCC and 64 KA were arranged in a tissue microarray (TMA) and stained for immunologic cell‐markers CD3, CD20 and CD68 as well as for proteins considered of relevance in tumorgenesis, namely NFκB/p65, IκB‐α, STAT3, p53, TRAP‐1, pRB, phosphorylated pRb, Cyld, p21, p16INK4, Survivin, Bcl‐xL, Caspase 3, Bak, FLK‐1/VEGF‐r2 and Ki‐67. In addition, the tumors were tested for presence of human papillomavirus by PCR. We detected that the two lesions differed significantly in expression of Bcl‐xL which was present in 84% of the SCC compared with only 15% in the KA (p < 0.001). The lower expression of the antiapoptotic protein Bcl‐xL in KA is consistent with a possible role of apoptosis in the regression of KA.

Sarcomatoid conversion of clear cell renal cell carcinoma in relation to epithelial-to-mesenchymal transition.

Approximately 8% of clear cell renal cell carcinoma cases contain regions of radically different morphology, demonstrating a mesenchymal appearance histologically resembling sarcomas. These biphasic neoplasms are called sarcomatoid clear cell renal cell carcinoma. Patients diagnosed with sarcomatoid clear cell renal cell carcinoma face a considerably worse prognosis due to an increased propensity for metastasis. In the present study we investigate whether the sarcomatoid conversion of clear cell renal cell carcinoma could be interpreted as linked to the process of epithelial-mesenchymal transition. Using 6 biphasic clear cell renal cell carcinoma cases we show that sarcomatoid clear cell renal cell carcinoma shares characteristic markers associated with loss of von Hippel-Lindau tumor suppressor with conventional clear cell renal cell carcinoma and also exhibits a markedly higher proliferative index. Furthermore the sarcomatoid elements demonstrate an enhanced expression of epithelial-mesenchymal transition related mesenchymal markers as compared with the clear cell renal cell carcinoma counterparts. We further selected a representative case, clinically demonstrating direct overgrowth of the sarcomatoid component into the liver and colon, for extended immunohistochemical characterization, resulting in a further set of positive and negative epithelial-mesenchymal transition markers as well as pronounced transforming growth factor β positivity, indicating that sarcomatoid clear cell renal cell carcinoma may be associated to epithelial-mesenchymal transition. Transforming growth factor β1 exposure of in vitro cultured primary clear cell renal cell carcinoma cells resulted in cells adopting a mesenchymal morphology similar to sarcomatoid clear cell renal cell carcinoma. Corresponding changes in RNA levels for key epithelial-mesenchymal transition markers were also seen. We therefore suggest that sarcomatoid clear cell renal cell carcinoma morphologically and immunohistochemically may represent a completed epithelial-mesenchymal transition and that transforming growth factor β1 could be an important driving force during the sarcomatoid transdifferentiation of clear cell renal cell carcinoma.

Tumors with non-functional RB1 are killed by reduced gamma-tubulin levels.

Background: γ-Tubulin moderates the expression of E2F-regulated promoters by direct binding to DNA. Results: RB1 and γ-tubulin proteins moderate each others expression by binding to their respective gene promoters. Conclusion: Reduction of γ-tubulin protein levels in tumors with nonfunctional RB1 leads to induction of apoptosis. Significance: The RB1/γ-tubulin signal network can be considered as a new target for cancer treatment. In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each others expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.