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Immune checkpoint inhibitors targeting the programmed cell death-1 receptor (PD-1) have been shown to improve the survival of patients with clear cell renal cell carcinoma (CCRCC). However, a benefit for CCCRC patients was observed with the FDA-approved PD-1 inhibitor Nivolumab irrespective of PD-1 ligand (PD-L1) expression status indicating there are potentially additional CCRCC checkpoint molecules worth exploring for targeted therapy. We therefore analyzed RNAseq data (n=534, CCRCCs; n=72, normal kidney specimens) from the TCGA project, and found that PD-L1 was not upregulated in CCRCC when compared to normal tissue whereas other checkpoint members i.e. CD300A and LAIR1 were among the most significantly upregulated genes in CCRCCs. In terms of overall survival, high expression of PD-L1 was associated with better outcome in CCRCCs while high expression of CD300A and LAIR1 were significantly associated with worse outcomes. We confirmed these findings in two independent cohorts of CCRCC patients. Furthermore, we performed cell type enrichment analysis, which showed that CD300A and LAIR1 are most likely expressed by tumor-associated macrophages in the CCRCC tumor microenvironment. Our results suggest that PD1/PDL1 might be suboptimal targets in CCRCC and that there are additional unexplored immune checkpoint molecules that warrant our attention. Note: This abstract was not presented at the meeting. Citation Format: Jonas Sjolund, David Lindgren, Helén Nilsson, Martin E. Johansson, Hakan Axelson, Kristian Pietras. Immune checkpoint inhibitors CD300A and LAIR1 are associated with poor prognosis in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1086.

Background : Studies in experimental models have implied perivascular cells (PC) as regulators of multiple aspects of tumor biology, including tumor growth, metastasis, immune cell activity and response to treatment. However, the extent of inter- and intra-case variability of perivascular cells in clinical samples, and potential of perivascular cells as biomarkers, remain poorly characterized. Methods: We have developed an integrated analytical pipeline to investigate vascular and PC status in clinical samples. The analytical procedure uses double-staining with one endothelial cell marker (CD34) and one of four PC markers; platelet-derived growth factor receptor-alpha and -beta (PDGFR-α, PDGFR-β;), smooth muscle α actin (ASMA) and desmin. Digital image-analyses are subsequently used to quantitate PC-related metrics including intensity of marker expression in perivascular areas, and fraction of marker-covered vessels. The approach has been applied to multiple cohorts of clinically well-annotated tumor collections from three cancer types; colorectal cancer (CRC), ovarian cancer (OC) and renal cell cancer (RCC). Results: Analyses of PC status uncovered previously un- recognized independent and heterogeneous expression of the analyzed perivascular markers on different levels (perivascular cells, vessels and tumors). Notably, expression of perivascular markers was largely independent of vessel size and density in all three cancer types. Comparative analyses across tumor types identified differences including e.g. higher abundance of PDGFR-β; positive perivascular cells in CRC. A comparison of perivascular status in matched primary tumors and distant metastases uncovered a large degree of dis-concordance of most vascular characteristics except perivascular PDGFR-β; (CRC and OC cohorts). Importantly, this finding implies that vascular status in metastases cannot, in general, be predicted by analyses of primary tumors. Efforts to uncover the basis for inter-case variations in PC status of CRC revealed strong associations between tumor PDGFR-β; PC status and both PC status in adjacent normal tissue and BRAF status, but not RAS mutations. These findings suggest both host features and cancer mutations as determinants of PC characteristics. Concerning relationships between PC features and survival, we observed significant associations between high perivascular PDGFR-β; status and shorter survival in OC and RCC. These associations remain significant in multivariate analyses including standard clinicopathological characteristics. Impact on response to chemotherapy (CT) treatment was analyzed in both early stage and metastatic CRC. Ongoing analyses indicate that low perivascular PDGFR-βdefines a sub-group with shorter survival in the CT-treated group. Explorative analyses of a small set of bevacizumab-treated metastatic CRC also demonstrated significantly shorter progression free survival in the subset with low perivascular PDGFR-β expression. Conclusion: Digital-image-analyses-supported quantifications of PC features in three different tumor types have revealed heterogeneous expression of PC markers in a manner independent of vessel density and vessel size. Host characteristics and oncogenic status have been identified as candidate determinants of perivascular PDGFR-β; status in CRC. Notably, perivascular PDGFR-β; was identified as a novel independent predictor of survival in OC, RCC and CRC. Treatment-stratified analyses in CRC also implied perivascular PDGFR-β; as a determinant of benefit of chemotherapy and VEGF-directed anti-angiogenic drugs. Citation Format: Sara corvigno, artur mezheyeuski, Magnus Frodin, Maja Bradic Lindh, Tormod Kyrre Guren, Per Pfeiffer, H. Elin Kure, Tone Ikdahl, Eva Skovlund, Kjell Magne Tveit, Kristian Pietras, Anna Portyanko, Ina Hrynchyk, David Edler, Anca Dragomir, friedrik Ponten, Jan Mulder, Camilla Qvortrup, Maria Hallström, Katarina Öhrling, G. Bea A. Wisman, Elisabeth Åvall-Lundqvist, Martin Johansson, Ulrika Harmenberg, Hans Nijman, Per Sandström, Hanna Dahlstrand, Halfdan Sorbye, Bengt Gimelius, Ate Van Der Zee, Lars Egevad, Arne Östman. Marker-defined perivascular cells predict prognosis and response to treatment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C38.

Background: Androgen ablation remains the most effective therapy for patients with advanced disease. Unfortunately, most patients progress to castrate resistant prostate cancer (CRPC) characterized with hyper-activation of the androgen receptor (AR). Enzalutamide, a potent AR inhibitor showed efficacy by prolonging survival in CRPC patients. However, ENZ resistance (ENZR) rapidly occurs in patients and in our pre-clinical model targeting AR in ENZ resistant tumors with a 3rd generation AR inhibitor is short lived. The role of signal transducer and activator of transcription (STAT) 3 in the progression of prostate cancer is well established and has been repeatedly linked with maintenance of a stem cell phenotype across cancers. Additionally, drug resistance has been hypothesized to occur via enrichment of cancer stem-like cells (CSC), a phenotype associated with poor survival in patients. Therefore, we propose a shift of focus to target CSC phenotype using small molecule inhibitor of STAT3 called GPA500, instead of the AR axis to deal with ENZ resistance. Methods and Results: We developed a unique model of ENZ-resistance and found that cell lines derived from serially passaged ENZR tumors displayed broad genetic diversity and differential AR activity. Notably, the cell lines 42DENZR and 42FENZR are PSAlow, harbor an expanded CSC population and have STAT3 hyper-activation compared to CRPC controls measured by STAT3-luc reporter. Accordingly, Crystal Violet and MTT proliferation assays showed that 42DENZR and 42FENZR cell lines were more sensitive to the STAT3 inhibitor GPA500 compared to CRPC control. Targeting the ENZ-R cells with GPA500 reduced mRNA levels of CD133, CD44, SOX2, OCT4 and Nanog. The reduction of these CSC markers was accompanied by reduced self-renewal capacity measured by spheroid assay. Cytometry analysis revealed that treatment with GPA500 reduced α2β1+, CD44+ and CD133+ (CSC) population in the 42D and 42F cells. Conclusion: In this study, we provide pre-clinical proof that targeting the STAT3 pathway using GPA500 in ENZ resistance as well as CRPC reduces cell proliferation as well as expression of stem cell markers. Impact: Exploring mechanisms of ENZR resistance serves a critical unmet need in PCa oncology, as the number of men with ENZ resistant CRPC continues to rise. Targeting STAT3 with the small molecule inhibitor GPA500 may provide an effective method to treat ENZR patients or delay the emergence of ENZ resistance in CRPC by reducing the emergence of stem cells. Citation Format: Sepideh Vahid, Daksh Thaper, Alistair Davies, Micaela Janse Van Rensburg, Kate Gibson, Krisi Ketola, Martin Johansson, Jennifer L. Bishop, Amina Zoubeidi. Galiellalactone derivative targets stem cell population in ENZ-resistant prostate cancer through inhibition of STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3342.

Clear cell renal cell carcinoma, ccRCC, is the most prevalent type of kidney cancer. In over 90% of ccRCCs the tumor suppressor protein von Hippel Lindau is inactivated, leading to a pseudohypoxic phenotype with vast effects on angiogenesis as well as energy metabolism. In accordance with the Warburg effect, aerobic glycolysis increases, while mitochondrial oxidative phosphorylation is decreased. We have previously published data showing that also the number and activity of mitochondria is strikingly reduced in primary human ccRCC cells compared to normal kidney epithelial cells. This was not seen in any of the established RCC cell lines included in the study. We are now continuing these studies by investigating the regulation of mitochondrial biogenesis in primary human ccRCC cells, and how the number and activity of mitochondria influences the tumorigenic properties of these cells. RNA has been isolated from ccRCC tissue areas identified as mitochondria high or low. The expression profile in these samples will be analyzed by RNA sequencing. Human primary ccRCC cells obtained from patient nephrectomies are kept in culture and the regulation of mitochondrial biogenesis in these cells is investigated. How the mitochondrial content affects tumorigenic properties such as proliferation, migration and resistance to apoptosis is also investigated. Preliminary data indicate that primary ccRCC cells have an absent or severly limited capability to increase the number of mitochondria on metabolic demand, and that the regulation of mitogenesis is defective. These results point towards a malfunctioning energy sensing and mitochondrial biogenesis in ccRCC cells that might be part of the explanation to the very low mitochondrial content in these cells. Citation Format: Helén Å. Nilsson, Martin E. Johansson. Regulation of mitochondrial biogenesis in primary human clear cell renal cell carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 207.

Background: The transcription factor Signal Transducer and Activator of Transcription Factor 3 (STAT3) is implicated in acquired drug resistance, metastases and immune suppression in prostate cancer and is a potential target in drug-resistant prostate cancer. Today, the treatment options for metastatic drug-resistant prostate cancer are very limited and existing immunotherapies have so far shown low efficacy. Cancer cells may avoid immune responses by expressing immunosuppressive markers or activating immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) play a major role in the suppression of antitumor immunity and active STAT3 signaling has been shown to be involved in the immunosuppressive functions of these cells. Elevated levels of MDSC have been found in the peripheral blood and at the tumor site of prostate cancer patients and to correlate with disease progression. Aims: In this study we aimed to investigate if human monocytes could be induced to differentiate into immunosuppressive cells in the presence of prostate cancer cells and if this effect could be blocked by the STAT3 inhibitor GPA500 (galiellalactone). Methods: Monocytes were isolated from peripheral blood mononuclear cells from healthy human donors using a magnetic monocyte enrichment kit. Monocytes were co-cultured with the prostate cancer cell lines DU145, LNCaP or longterm IL6 treated LNCaP cells (LNCaP-IL6+) in transwells allowing cancer cells and monocytes to share culture medium. The co-cultures were grown for three days with or without the STAT3 inhibitor GPA500 (5 uM or 10 uM) with subsequent analysis by flow cytometry for cell surface markers of MDSC differentiation (CD14+ HLA-DRlo). The presence of active STAT3 (pSTAT3) in the prostate cancer cell lines was determined by Western blot. Results: Monocytes in the presence of DU145 and LNCaP-IL6+ cells showed a significantly increased population of CD14+ HLA-DRlo cells. In the presence of GPA500 the induction of this population was inhibited in a dose dependent manner compared to untreated cells. Co-culture with LNCaP cells that lack pSTAT3 did not induce this population in monocytes after three days in culture. Furthermore, GPA500 induced a dendritic cell-like population in monocytes in the absence of prostate cancer cell co-culture. This population was also observed in monocytes when co-cultured with LNCaP-IL6+ cells, but not in DU145 co-cultures, with GPA500 present. Conclusion: The prostate cancer cell lines DU145 and LNCaP-IL6+ were able to induce a monocyte population displaying MDSC markers and this induction could be inhibited by the STAT3 inhibitor GPA500. Impact: This study demonstrates that inhibiting the transcription factor STAT3 in prostate cancer could potentially reverse the immunosuppressive mechanisms caused by MDSC activation and that STAT3 inhibitors, alone or in combination with other therapies, could be a new treatment option for prostate cancer. Citation Format: Rebecka Hellsten, Karin Leandersson, Anders Bjartell, Martin E. Johansson. Prostate cancer cell-induced differentiation of human monocytes into MDSCs ex vivo is inhibited by targeting STAT3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 555.

Background: The role of signal transducer and activator of transcription (STAT) 3 in the progression of Prostate Cancer (PCa), the most common male cancer in North America, is well established. Although androgen ablation remains the most effective therapy for patients with advanced disease, most progress to castrate resistant prostate cancer (CRPC). Despite the development of new potent anti-androgens, like Enzalutamide (ENZ), which prolongs survival in CRPC patients, ENZ resistance rapidly occurs. Re-activation of the androgen receptor (AR) is a hallmark of CRPC and can occur via different mechanisms, including STAT3 activation. Furthermore, STAT3 can promote CRPC independently of AR activity, however whether STAT3 promotes resistance to ENZ in CRPC remains unknown. We have found that our LNCaP xenograft derived ENZR cell lines exhibit higher activity of the STAT3 pathway compared to CRPC. Additionally, in this model further targeting the AR using novel anti-androgens is short lived or ineffective. Therefore, we propose that targeting STAT3 using the small molecule inhibitor GPA500, alone or in combination with anti-androgens may delay or treat ENZR CRPC. Methods and Results: We developed a unique model of ENZ-resistance and found that cell lines derived from serially passaged ENZR tumors displayed broad genetic diversity and differential AR activity but consistent STAT3 hyper-activation compared to CRPC controls. WB analysis showed that all ENZR cell lines had increased pSTAT3Y705 compared to CRPC. Accordingly, Crystal Violet and MTT proliferation assays showed that ENZR cell lines were more sensitive to the STAT3 inhibitor GPA500 compared to CRPC control. The reduction in growth of GPA500 treated ENZR cells was independent of PARP cleavage, whereas GPA500 induced PARP cleavage in CRPC cells. Moreover, in ENZR cell lines with high AR activity and CRPC control cells, inhibition of STAT3 reduced AR activity as shown with WB and qRT-PCR for AR downstream targets like PSA and an AR-luciferase reporter assay. In ENZR cells with low AR activity and a stem cell phenotype, GPA500 treatment reduced surface expression of α2β1, CD44 and CD133. Inhibition of STAT3 activity was confirmed by WB as well as qRT-PCR analysis of STAT3 regulated genes like c-Myc, MCL1, BCL-XL. Conclusion: In this study, we provide pre-clinical proof that targeting the STAT3 pathway using GPA500 in ENZ resistance as well as CRPC reduces cell proliferation and AR activity as well as expression of stem cell markers. Impact: Exploring mechanisms of ENZR resistance serves a critical unmet need in PCa oncology, as the number of men with ENZ resistant CRPC continues to rise. Targeting STAT3 with the small molecule inhibitor GPA500 may provide an effective method to treat ENZR patients or delay the emergence of ENZ resistance in CRPC via reduction of AR activity and/or by suppressing aggressive tumor cell phenotypes such as cancer stem cells. Citation Format: Daksh Thaper, Sepideh Vahid, Jennifer L. Bishop, Martin Johansson, Amina Zoubeidi. New derivative of galiellalactone inhibits the STAT3 activity and suppresses ENZ-resistant Prostate Cancer in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2015-728

The proximal tubular segments of the kidney are extremely energy demanding, rendering this epithelium exceptionally sensitive to ischemic insults. Acute renal injury is a potentially life threatening condition, where the tubular cells detach and die. However, the tubules of injured kidneys have a substantial regenerative ability, and epithelial continuity, can with proper supportive care be restored. The cellular source of regeneration has been debated and until now the proximal tubules has been considered a completely homogenous cellular epithelium. Using an assay based on aldehyde dehydrogenase (ALDH) activity followed by FACS, we have recently discovered a cell with progenitor cell characteristics scattered within the proximal tubules of the kidney. These novel proximal tubule rare cells (PTRCs) expressed stem cell markers CD133 and CD24, as well as intermediate filaments not normally seen in PTs. The gene expression profile of these cells shows a striking resemblance to that of papillary renal cell carcinoma (pRCC) and the gene signatures are also associated with a worse prognosis in clear cell renal carcinoma. Additionally, cells positive for PTRC-markers were seen in regenerating tubules after acute kidney injury. We have characterized these cells further at an ultra structural level, using electron microscopy. The cells were small, flask shaped and had a more densely packed chromatin pattern and drastically fewer mitochondria than the surrounding proximal tubular cells, indicating a different metabolic state. We suggest that these cells, in contrast to the surrounding highly oxygen dependent proximal tubular cells, constitute renal reserve cell pool capable of surviving ischemic renal injury with a capacity to spearhead subsequent tubular repopulation. We suggest that the increased probability for survival of these cells is due to their low mitochondrial content, structural robustness and anti-apoptotic signaling. Also, due to the considerable transcriptional similarities between pRCC, renal adenomas and the PTRCs we hypothesize that pRCC may arise through multistep oncogenic transformation of PTRCs. Citation Format: Jennifer Hansson, Kjell Hultenby, Catharina Cramnert, David Lindgren, Håkan Axelson, Martin E. Johansson. Characterization of a novel cell type in human renal proximal tubules with connection to renal cell carcinoma development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2013-5010

Renal cancer causes over 100,000 annual deaths worldwide and constitutes about 3% of all solid cancers. The majority of renal cell carcinomas (85%), referred to as clear cell renal cell carcinoma (CCRCC), are notorious for their metastatic frequency and high resistance to conventional cancer therapies. Several new targeted therapies have been approved for treatment of metastatic renal cell carcinoma (mRCC), and although they induce a longer progression free survival, the effect on overall survival has not been statistically significant. One third of the patients with CCRCC present with advanced disease at diagnosis, and another third will develop metastases eventually, even if the original tumor is successfully removed. A metastatic form of the disease correlates with a bad poor prognosis reflected in the 5-year survival, which is less than 11% for this patient group. A fungal toxin from the Cortinarius-family, NC-001, have been shown to be specifically toxic to the proximal tubular cells in the kidney. We have shown that this sensitivity also is extended to renal cancer cells which have evolved from these. Preliminary data demonstrate that NC-001 reduces viability in five renal cancer cell lines, originating both from primary tumors and metastatic lesions. HUVEC cells seem unaffected, however at the highest dose (800µM) a reduction in viability is observed. Presently, we are evaluating the molecular mechanism behind the effect of NC-001. In vitro results show an induction of apoptosis via the intrinsic pathway, by activation of caspase-8 and further downstream by caspase-3. Caspase-9 seems to bewas unaffected. FACS analysis with Alexa Flour488 conjugated Annexin V and phosphatidylserine (PI) of SKRC-52 cells treated with 400µM for 24 hours, shows increased apoptosis and necrosis. A xenograft model of CCRCC, in radiated athymic rats, was developed to further investigate NC-001's effects in vivo. Since NC-001 affects the proximal tubular cells in the kidney and induces severe renal failure, an automated peritoneal dialysis system for rats was developed as renal replacement therapy. The rats were treated via the dialysis solution containing 40µM NC-001 for two days. Six days later, extensive necrosis was evident in the tumors of treated rats (n=5) compared to controls (n=5). In summary, our results indicate that NC-001 induces apoptosis, mainly via the intrinsic pathway, and necrosis in SKRC-52 cells and also in solid CCRCC xenograft tumors. NC-001 has therefore potential as a curative treatment of metastatic CCRCC. The sole expected side effect of NC-001, based on hundreds of case reports of Cortinarius intoxications in the literature, is collateral loss of kidney function. While this may appear dramatic, it is surely preferable to death from metastatic cancer, especially if long term survival can be achieved. Besides, it can be well managed with dialysis, and later on, kidney transplantation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-118. doi:1538-7445.AM2012-LB-118

About 8% of clear cell renal cell carcinoma (CCRCC) cases contain areas of a radically different morphology, demonstrating spindle shaped cells and a histological resemblance to sarcomas. These biphasic neoplasms are called sarcomatoid CCRCC (sCCRCC). Patients diagnosed with sCCRCC face a considerably worse prognosis due to an increased propensity for metastasis. Using a set of biphasic CCRCC cases, we showed that sCCRCC has a higher proliferative index and more prononced vimentin expression as compared to the CCRCC counterpart. We selected a CCRCC/sCCRCC case demonstrating overgrowth as well as metastatic spread for extended immunohistochemical characterization. Using mouse xenografts we evaluated a $$$$CCRCC cell-line and put forward the hypothesis that some CCRCC cell-lines may be derived from the sarcomatoid component rather than from the clear cell part, with obvious implications for the validity of in vitro models of CCRCC. We confirm that sCCRCC is derived from the clear cell component and show that sCCRCC morphologically and immunohistochemically may represent a completed epithelial mesenchymal transition (EMT). Furthermore we suggest that TGF-β1 may be the driving force behind sarcomatoid transdifferentiation i.e. EMT in CCRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3439. doi:10.1158/1538-7445.AM2011-3439

Small cell lung carcinoma (SCLC) is an extremely aggressive tumor that is widely metastasized in early stages of the disease and the 5-year survival rate for these patients is only approximately 9-15%. Here we study the adaptive response of SCLC cells to lower oxygen levels, since tumor hypoxia (∼1% oxygen) is related to aggressive tumor behaviour and poor outcome in several tumor forms. Areas of hypoxia are a common characteristic in solid tumors and in response to hypoxia tumor cells induce expression of several genes involved in a variety of processes. This adaptive response is primarily regulated by the transcription factors hypoxia inducible factor (HIF)-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others lung adenocarcinoma, neuroblastoma and breast carcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated the viability of SCLC cells cultured at hypoxia and characterized the expression levels of HIF transcription factors in SCLC cells and specimens using quantitative real-time PCR, western blot and immunohistochemistry. Expression levels of well known hypoxia-driven genes were studied and to further characterize the hypoxic adaptive response, a gene expression profile of hypoxic SCLC cells was performed using microarray analyses. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Our results show that SCLC cells have a high adaptive capacity to hypoxia, even though a modest induction of well known hypoxia-driven genes is observed. Interestingly, the HIF-1α and HIF-2α are differentially regulated in SCLC cells as compared to non-SCLC. In SCLC cells, no HIF-2α protein is detectable whereas HIF-1α protein levels are sustained and increased up to 96 hours. In addition, knock-down of HIF-1α at 0.1% oxygen are not affecting the viability of the SCLC cells and our gene expression profile of hypoxic SCLC cells has identified genes important for SCLC cells survival at hypoxia. Our data suggest that SCLC cells have an adaptive response to hypoxia that is partially HIF-independent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2011-2058

Here we study the adaptive capacity of small cell lung carcinoma (SCLC) cells to hypoxia and the importance of activation of hypoxia-inducible factor (HIF) transcription factors in these cells. SCLC is an extremely aggressive tumor form and the 5-year survival rate for these patients is only approximately 9-15%. Regions of hypoxia (∼1% oxygen) often appear in solid tumors and are associated with aggressive tumor behaviour and poor outcome in several tumor forms. Tumor cells adapt to a hypoxic environment by changing the transcription of genes involved in a variety of processes, like angiogenesis, cell survival, and metabolism. This adaptive response is primarily regulated by the transcription factors HIF-1α and HIF-2α, which become stable at low oxygen levels. Studies using among others neuroblastoma, breast carcinoma and lung adenocarcinoma cells have demonstrated that HIF-1α primarily mediates the acute hypoxic response, whereas HIF-2α dominates during the more chronic phase of hypoxia. We investigated cell survival in a panel of SCLC cell lines cultured at different oxygen pressures. In these cells we also studied expression levels of well known hypoxia-driven genes using quantitative real-time PCR. Quantitative real-time PCR, Western blot and immunohistochemistry were used to characterise the expression of HIF transcription factors in SCLC cells as well as in SCLC specimens. In all experiments a panel of non-SCLC cells and neuroblastoma cells were included for comparison. Since tumor hypoxia correlates with poor prognosis it is important to elucidate how tumor cells adapt in response to hypoxia. Our results show that SCLC cells survive well at hypoxia, although the proliferation is somewhat reduced with lower oxygen pressure. Even though the SCLC cells have a high adaptive capacity, only a moderate up-regulation was observed for the investigated hypoxia-regulated genes at 1% oxygen. In addition, the expression patterns of HIF-1α and HIF-2α differ in SCLC as compared to non-SCLC, indicating that a “typical” adaptive response to hypoxia does not occur in SCLC cells. Based on these results a gene expression profile of hypoxic SCLC cells using microarray analyses are performed to further characterise the hypoxic adaptive response. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 452.

Contemporary tumor biology challenges the previous belief that all cancer cells are capable of sustaining tumor growth. Instead, data is accumulating in favor of a concept where a small percentage of the total tumor cell number, are believed to have the capacity to drive cancerous growth. These cells are called tumor initiating cells or cancer stem cells (CSCs). The presence of CSCs in the tumor may increase malignancy potential, mediate resistance to conventional therapy and lead to poor survival outcomes. To achieve effective prognostic and therapeutic strategies it is important to identify markers for and CSC-unique properties. Our study aims at identifying cells with stem cell like properties in renal cell carcinoma (RCC) and specify their role in tumorigenesis. ALDH activity has been used to isolate hematopoetic and breast stem cells and high expression of ALDH has been identified in breast cancer with poor prognosis. We have used the ALDEFLUOR assay to isolate stem cell like cells from both primary and established RCC cultures. We found that the ALDH positive cells have a greater colony forming capacity and ability to grow under low attachment conditions, as compared to cells with low ALDH activity. We also found that CD133, another known stem cell marker, was expressed at higher levels in the ALDH positive cells. The ALDH positive cells were characterized using global gene expression analysis, producing a framework for further characterization. The clinical validity of this population was assessed using gene expression from a data set of 176 primary RCCs and these analysis indicate that the stem cell expression profile is associated with decreased disease specific survival. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3368.

Introduction: Tumor stem cells have shown resistance to conventional cancer therapy and may be a cause for recurrent cancer, making tumor stem cell targeted therapy attractive. The fungal metabolite galiellalactone is suggested to be a Stat3 specific inhibitor and we have previously shown that galiellalactone inhibits prostate cancer (PCa) cell growth, both in vitro and in vivo, in PCa cells expressing phosphorylated Stat3 (Hellsten et al, 2008). Aldehyde dehydrogenase (ALDH) has shown promise as a marker of cancer stem cells and in this study we investigated the ALDH activity in human PCa cell lines and the effects of galiellalactone on ALDH+ PCa cells. Methods: The human PCa cell lines DU145, LNCaP, and long-term interleukin-6 stimulated LNCaP (LNCaP-IL6) cells were used. Galiellalactone was prepared by synthesis (Johansson & Sterner, 2002). PCa cells were subjected to Aldefluor assay followed by flow cytometry to detect ALDH+ subpopulations. WST-1 proliferation assay was performed to study the effect of galiellalactone on proliferation of ALDH+ and ALDH- cells sorted from LNCaP-IL6 cells. Results: In DU145 cells 2 ± 0.25% of the cells were ALDH+ and in LNCaP-IL6 cells, 2 ± 0.36% were ALDH+. No ALDH+ cells were detected in LNCaP cells. Treatment with 5, 10, 25 or 50 µM galiellalactone for 24h decreased the ALDH+ cell population in DU145 and LNCaP-IL6 cells in a dose dependent manner: 25 µM galiellalactone decreased the ALDH+ population in DU145 cells by 40% and by 75% in LNCaP-IL6 cells. In LNCaP-IL6 cells a dose dependent decrease in ALDH+ cells was observed after 72h (65% decrease at 25 µM galiellalactone). However, in DU145 cells no difference in the relative amount of ALDH+ cells was observed in galiellalactone treated DU145 cells compared to untreated control cells after 72h. The proliferation was decreased by galiellalactone in a dose dependent manner in all cell populations isolated from LNCaP-IL6 cells. The proliferation of ALDH+ cells isolated from LNCaP-IL6 cells was inhibited by 75%, ALDH- cells by 75% and unsorted LNCaP-IL6 cells by 80% after treatment with 25 µM galiellalactone for 48h. Conclusions: ALDH+ subpopulations, possibly representing tumor stem cell populations were detected in human PCa cell lines, and were clearly inhibited by galiellalactone. ALDH is associated with cell growth and drug resistance, and targeting ALDH+ PCa cells with galiellalactone may be a novel potent treatment against therapy resistant PCa. The effect and mechanism of galiellalactone treatment of ALDH+ PCa cells will be further investigated. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3554.