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   Biotech / MedicalNanoViricides - Nanobiotechnology


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From: donpat2/13/2019 8:57:09 AM
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Microbes and Me

Could gut bacteria microbes make you fat?



A varied biome of gut bacteria is thought to have several benefits, even so far as improving our moods.

New evidence suggests it could also have a physical effect – making you skinnier, or even heavier.

bbc.com

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To: donpat who wrote (1189)2/18/2019 1:56:45 PM
From: donpat
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Oncotarget. 2017 Sep 15; 8(40): 68095–68107.
Published online 2017 Jul 28. doi: 10.18632/oncotarget.19677

PMCID: PMC5620239
PMID: 28978099

Mitochondrial markers predict survival and progression in non-small cell lung cancer (NSCLC) patients: Use as companion diagnostics

Federica Sotgia1 and Michael P. Lisanti1

Author information Article notes Copyright and License information Disclaimer

This article has been cited by other articles in PMC.

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Abstract
Here, we used an informatics-based approach to identify novel biomarkers of overall survival and tumor progression in non-small cell lung cancer (NSCLC) patients. We determined whether nuclear-encoded genes associated with mitochondrial biogenesis and function can be used to effectively predict clinical outcome in lung cancer. This strategy allowed us to directly provide in silico validation of the prognostic value of these mitochondrial components in large, clinically-relevant, lung cancer patient populations. Towards this end, we used a group of 726 lung cancer patients, with negative surgical margins. Importantly, in this group of cancer patients, markers of cell proliferation (Ki67 and PCNA) were associated with poor overall survival, as would be expected. Similarly, key markers of inflammation (CD163 and CD68) also predicted poor clinical outcome in this patient population. Using this approach, we identified >180 new individual mitochondrial gene probes that effectively predicted significantly reduced overall survival, with hazard-ratios (HR) of up to 4.89 (p<1.0e-16). These nuclear-encoded mitochondrial genes included chaperones, membrane proteins as well as ribosomal proteins (MRPs) and components of the OXPHOS (I-V) complexes. In this analysis, HSPD1, a key marker of mitochondrial biogenesis, had the highest predictive value and was also effective in predicting tumor progression in both smokers and non-smokers alike. In fact, it had even higher predictive value in non-smokers (HR=5.9; p=3.9e-07). Based on this analysis, we conclude that mitochondrial biogenesis should be considered as a new therapeutic target, for the more effective treatment of human lung cancers. The mitochondrial biomarkers that we have identified could serve as new companion diagnostics to assist clinicians in more accurately predicting clinical outcomes in lung cancer patients, driving more personalized cancer therapy.

Keywords: lung cancer, mitochondrial biomarkers, treatment failure, relapse, recurrence

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INTRODUCTIONTreatment failure is the most critical obstacle for more effective anti-cancer therapy and personalized medicine [ 1, 2]. As such, this still dramatically limits the efficacy of most cancer treatments, especially in lung cancer patients. As a consequence, better biomarkers are needed for the early stratification of lung cancer patients into low-risk and high-risk groups at diagnosis [ 13].

Here, we examined the hypothesis that markers of mitochondrial biogenesis and function may have significant prognostic value in the early identification of high-risk lung cancer patients, with poor overall clinical survival and tumor progression. In this context, we employed a bioinformatics approach to assess the possible utility of nuclear-encoded mitochondrial gene transcripts in predicting clinical outcome.

Our results indicate that > 180 different mitochondrial gene probes can be used individually, to predict poor overall survival in lung cancer patients. As such, we discuss the possibility that mitochondria should be therapeutically targeted, to improve the effectiveness of current lung cancer therapy and overall survival.

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RESULTS
Value of proliferative and inflammatory markers in the patient populationTo identify new potential biomarkers, here we used publically available transcriptional profiling data from the tumors of lung cancer patients, with negative surgical margins (Figure ?(Figure1),1), with 10 years of follow-up. Since proliferative markers are used as primary endpoints in clinical trials, we first assessed the prognostic value of Ki67 and PCNA, in this patient population. Tables ?Tables1,1, ?,22 and Figure ?Figure2A2A both show the prognostic value of these markers. The hazard-ratios for Ki67 and PCNA were 4.85 and 1.82, respectively, for overall survival (OS).





Figure 1
Diagram showing our bio-informatics approach to lung cancer biomarker discoveryFor this analysis, we chose to focus on non-small lung cancer patients, with negative surgical margins, and 10-years of follow-up data (N = 726). In this context, we evaluated the prognostic value of mitochondrial markers for predicting overall survival, time to first progression, and post-progression survival.



Table 1
Prognostic Value of KI67 in Lung Cancer
Gene Probe IDSymbolHazard-RatioLog-Rank Test
212020_s_atMKI674.852.2e-16
212021_s_atMKI673.113.4e-11
212023_s_atMKI673.042.4e-12
212022_s_atMKI672.967.4e-14
Combined4.437.0e-14


Table 2
Prognostic Value of PCNA and Markers of Inflammation in Lung Cancer
Gene Probe IDSymbolHazard-RatioLog-Rank Test
217400_atPCNA1.824.1e-07
216233_atCD1631.955.6e-09
215049_x_atCD1631.390.006
203645_s_atCD1631.300.03
203507_atCD681.590.0002






Figure 2
Markers of proliferation and inflammation predict poor overall survival in high-risk lung cancer patientsWe assessed the predictive value of Ki67 and PCNA in N = 726 lung cancer patients, with negative surgical margins. A. Note that high transcript levels of Ki67 and PCNA are associated with significantly reduced overall survival. Please note that the official gene name for the Ki67 protein is MKI67. B. Note that that high transcript levels of CD163 and CD68 are associated with significantly reduced overall survival.



We also assessed the prognostic value of two macrophage-specific markers of inflammation. Table ?Table22 and Figure ?Figure2B2B show that CD163 and CD68 both effectively predict overall survival, with hazard-ratios of 1.95 and 1.59, respectively. Thus, conventional markers of proliferation and inflammation can be used to predict overall survival in lung cancer patients.

Value of individual mitochondrial markersTo test our hypothesis that increased mitochondrial mass, biogenesis and function contributes towards poor overall survival in lung cancer patients, we next assessed the prognostic value of specific mitochondrial markers.

Initially, we examined the behavior of mitochondrial chaperones and mitochondrial membrane proteins. Table ?Table33 and Figure ?Figure33 both show that HSP60 (HSPD1) has the best prognostic value, with a hazard-ratio of 4.89 (p < 1.0e-17). Members of the TIMM and TOMM gene families also had prognostic value; AKAP1 and SLC25A5 also had significant value. Similar results were also obtained with mitochondrial creatine kinase isoforms (HR = 2.88-to-1.51) and PRKDC (DNA-PK), a critical kinase that helps maintain the integrity and the copy number of the mitochondrial genome (mt-DNA) (HR = 4.69-to-1.65), which functions in the DNA damage response.

Table 3
Prognostic Value of Mitochondrial HSPs and Other Mitochondrial Proteins
Gene Probe IDSymbolHazard-RatioLog-Rank Test
HSPs and Membrane Proteins (28 probes in total)
200806_s_atHSPD14.89<1.0e-16
218119_atTIMM234.681.1e-16
218357_s_atTIMM8B4.267.8e-16
203342_atTIMM17B3.312.5e-11
203093_s_atTIMM442.291.1e-09
217981_s_atTIMM10B2.151.2e-06
218316_atTIMM92.064.3e-08
201821_s_atTIMM17A2.041.7e-09
218188_s_atTIMM131.948.5e-09
218118_s_atTIMM231.831.8e-07
218408_atTIMM101.794e-05
202264_s_atTOMM404.291.1e-14
217960_s_atTOMM223.191.3e-13
201870_atTOMM342.839.8e-12
201812_s_atTOMM72.845.4e-13
201512_s_atTOMM70A1.903.1e-08
212773_s_atTOMM201.540.0006
217139_atVDAC13.741.9e-14
217140_s_atVDAC12.581.1e-16
212038_s_atVDAC11.637.8e-05
208844_atVDAC33.643.9e-14
211662_s_atVDAC22.366e-14
210625_s_atAKAP11.881.3e-06
200657_atSLC25A51.540.0001
Mitochondrial Creatine Kinase (2 probes in total)
202712_s_atCKMT1A2.887.8e-10
205295_atCKMT21.510.0005
Mitochondrial Genome Maintenance (3 probes in total)
210543_s_atPRKDC4.691.1e-16
208694_atPRKDC2.234.3e-12
215757_atPRKDC1.654.0e-05

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Figure 3
HSPD1, mitochondrial membrane proteins and PRKDC are associated with poor clinical outcome in lung cancer patientsA. Note that that high transcript levels of HSPD1 and TIMM23 are associated with significantly reduced overall survival. B. Note that that high transcript levels of PRKDC are associated with significantly reduced overall survival.



Secondly, we examined the prognostic value of mitochondrial ribosomal proteins (MRPs), which contribute to the synthesis of key members of the OXPHOS-complexes, and are essential for mitochondrial biogenesis (Table ?(Table4).4). Twenty-one components of the large subunit (MRPLs) showed significant prognostic value, with hazard-ratios between 4.36 and 1.47. Notably, MRPL48 had the best prognostic value. Fifteen different components of the small subunit (MRPSs) showed significant prognostic value, with hazard-ratios between 4.10 and 1.27. As such, thirty-six different MRPs all predicted poor overall survival. Kaplan-Meier curves for representative examples are shown in Figure ?Figure4,4, panels A & B.

Table 4
Prognostic Value of Mitochondrial Ribosomal Proteins
Gene Probe IDSymbolHazard-RatioLog-Rank Test
Large Ribosomal Subunit (21 probes in total)
218281_atMRPL484.361.9e-15
213897_s_atMRPL233.555.4e-13
219162_s_atMRPL113.292.5e-13
221997_s_atMRPL523.203.6e-14
221692_s_atMRPL343.081.6e-11
203931_s_atMRPL122.823.3e-12
218887_atMRPL22.814.4e-11
217919_s_atMRPL422.541.6e-13
218270_atMRPL242.351.8e-09
218105_s_atMRPL42.321.6e-09
218202_x_atMRPL442.192.5e-10
222216_s_atMRPL172.021.4e-08
218890_x_atMRPL351.965.7e-09
204599_s_atMRPL281.911.4e-07
220527_atMRPL201.849.1e-05
201717_atMRPL491.688.7e-06
218049_s_atMRPL131.688.1e-06
217980_s_atMRPL161.661.5e-05
203152_atMRPL401.620.0001
218027_atMRPL151.590.0001
203781_atMRPL331.470.001
Small Ribosomal Subunit (19 probes in total)
204331_s_atMRPS124.101.1e-16
210008_s_atMRPS123.934.9e-14
204330_s_atMRPS123.271e-13
213840_s_atMRPS122.992.3e-12
217932_atMRPS73.552.3e-12
218001_atMRPS23.281e-11
221688_s_atMRPS43.097.7e-11
211595_s_atMRPS112.969.1e-12
215919_s_atMRPS111.550.0002
218112_atMRPS342.437.6e-08
212604_atMRPS312.292.7e-07
219819_s_atMRPS281.742.7e-06
217942_atMRPS351.708.4e-06
221437_s_atMRPS151.590.0001
12145_atMRPS271.617.4e-05
218398_atMRPS301.470.003
218654_s_atMRPS331.350.01
203800_s_atMRPS141.270.05

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Figure 4
Mitochondrial ribosomal proteins (MRPs) are associated with poor clinical outcome in lung cancer patientsA. Note that high transcript levels of MRPL48 and MRPL23 predict significantly reduced overall survival. B.Similarly, high transcript levels of MRPS12 and MRPS7 predict significantly reduced overall survival.



We also assessed the prognostic value of members of the OXPHOS complexes I-V. These results are summarized in Table ?Table5.5. Remarkably, 88 different gene probes for the OXPHOS complexes showed hazard-ratios between 4.46 and 1.39. COX5B (complex IV) had the best prognostic value (HR = 4.46; p = 5.3e-15). NDUFB3 (complex I) also showed significant prognostic value (HR = 4.30; p = 3.6e-15). Kaplan-Meier curves for members of complex I and II are shown in Figure 5A & 5B, while results with members of complex III and IV are shown in Figure 6A & 6B. Results with complex V are shown in Figure ?Figure77.

Table 5
Prognostic Value of Mitochondrial OXPHOS Complexes
Gene Probe IDSymbolHazard-RatioLog-Rank Test
Complex I (27 probes in total)
203371_s_atNDUFB34.303.6e-15
203189_s_atNDUFS84.154.4e-16
203190_atNDUFS82.942.1e-11
209303_atNDUFS43.831.1e-15
218484_atNDUFA4L23.332.1e-13
218226_s_atNDUFB43.211.8e-14
220864_s_atNDUFA133.009.5e-11
202941_atNDUFV23.001.3e-13
201740_atNDUFS32.921.2e-11
217860_atNDUFA102.773e-14
218563_atNDUFA32.231.9e-10
214241_atNDUFB82.231.5e-09
218201_atNDUFB22.211.2e-08
215850_s_atNDUFA51.833.6e-07
202785_atNDUFA71.813e-07
202298_atNDUFA11.723e-06
201966_atNDUFS21.706.6e-06
202839_s_atNDUFB71.640.0009
201757_atNDUFS51.644.3e-05
209224_s_atNDUFA21.596.6e-05
208969_atNDUFA91.560.0002
211752_s_atNDUFS71.500.0007
203613_s_atNDUFB61.490.0009
209223_atNDUFA21.490.0009
218320_s_atNDUFB111.480.001
218200_s_atNDUFB21.480.001
208714_atNDUFV11.440.002
Complex II (5 probes in total)
216591_s_atSDHC4.277.8e-16
202004_x_atSDHC3.644e-14
210131_x_atSDHC3.454.2e-14
202675_atSDHB2.067.4e-07
214166_atSDHB1.942.5e-08
Complex III (8 probes in total)
201568_atUQCR73.343.7e-13
209066_x_atUQCR62.962.5e-10
202233_s_atUQCR82.095.9e-07
208909_atUQCRFS11.692.6e-05
201066_atUQCR4/CYC11.540.0006
207618_s_atBCS1L1.540.0003
205849_s_atUQCR61.480.0008
202090_s_atUQCR1.450.004
Complex IV (19 probes in total)
211025_x_atCOX5B4.465.3e-15
202343_x_atCOX5B3.971.1e-16
213735_s_atCOX5B2.159.6e-10
213736_atCOX5B1.510.0015
200925_atCOX6A3.941.1e-16
201119_s_atCOX8A3.782.4e-15
203880_atCOX173.553.9e-15
201754_atCOX6C3.241.8e-14
217249_x_atCOX7A23.053.3e-13
201441_atCOX6B2.933.8e-12
206353_atCOX6A22.771.8e-11
203858_s_atCOX102.441.3e-09
202110_atCOX7B2.292.5e-12
216003_atCOX102.181.8e-07
221550_atCOX152.091.5e-10
217451_atCOX5A2.019e-06
218057_x_atCOX4NB1.540.0008
204570_atCOX7A1.510.0015
202698_x_atCOX4I11.390.01
Complex V (23 probes in total)
202961_s_atATP5J24.381.3e-14
207507_s_atATP5G34.14<1e-17
207508_atATP5G32.341.6e-13
210149_s_atATP5H3.703.7e-15
209492_x_atATP5I3.337.7e-13
207335_x_atATP5I2.142e-08
203926_x_atATP5D3.022.7e-11
213041_s_atATP5D2.413.1e-10
208764_s_atATP5G22.752.9e-10
207552_atATP5G22.554.3e-09
217368_atATP5G21.854.9e-07
217801_atATP5E2.622e-09
210453_x_atATP5L2.561.8e-11
207573_x_atATP5L2.251.9e-10
208746_x_atATP5L2.107.4e-10
201322_atATP5B1.881.5e-07
206992_s_atATP5S1.882.9e-07
206993_atATP5S1.852.1e-07
208972_s_atATP5G1.875.4e-08
221677_s_atATP5O1.716.8e-06
208870_x_atATP5C1.540.0008
205711_x_atATP5C1.420.004
213366_x_atATP5C1.400.007

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Figure 5
Mitochondrial complex I and II proteins are associated with poor clinical outcome in lung cancer patientsA. Note that high levels of NDUFB3 and NDUFS8 predict significantly reduced overall survival. B. Similarly, high levels of SDHC and SDHB predict significantly reduced overall survival.







Figure 6
Mitochondrial complex III and IV proteins are associated with poor clinical outcome in lung cancer patientsA. Note that high levels of UQCR7 and UQCR6 predict significantly reduced overall survival. B. Similarly, high levels of COX5B and COX6A predict significantly reduced overall survival.







Figure 7
Mitochondrial complex V proteins are associated with poor clinical outcome in lung cancer patientsNote that high levels of ATP5J2 and ATP5G3 predict significantly reduced overall survival.



Mitochondrial genes have predictive value in both “smoking” and “non-smoking” patient populations: overall survival and tumor progressionIn order to further test the prognostic power of these individual mitochondrial biomarkers, we next selected the most promising one, HSPD1, and assessed its ability to predict tumor progression in the whole patient population (N = 726). Importantly, Figure ?Figure88 shows that the levels of HSPD1 effectively predict time to tumor progression and post-progression survival, with hazard ratios of 3.28 and 1.88, respectively.





Figure 8
The mitochondrial chaperone, HSPD1, predicts tumor progression in lung cancer patientsNote that the levels HSPD1 effectively predict time to first progression (Left panel) and post-progression survival (Right panel).



A similar analysis was also carried out when the patient population was sub-divided into smokers (N = 464) and non-smokers (N = 160) (Figures ?(Figures99 and ?and10).10). Using this approach, HSPD1 showed increased prognostic power in the non-smoking patient population, reaching a hazard-ratio of 5.9 for overall survival; however, HSPD1 still retained its prognostic value in the smoking patient population (Figures ?(Figures9A9A and 10A).




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Figure 9
The mitochondrial chaperone, HSPD1, predicts poor clinical outcome and tumor progression in lung cancer patients: SmokersNote that the levels HSPD1 effectively predict overall survival A., as well as time to first progression and post-progression survival B., in the “smoking” patient population.






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Figure 10
The mitochondrial chaperone, HSPD1, predicts poor clinical outcome and tumor progression in lung cancer patients: Non-SmokersNote that the levels HSPD1 effectively predict overall survival A., as well as time to first progression and post-progression survival B., in the “non-smoking” patient population.



In this context, this trend was also true for tumor progression, as HSPD1 was a better predictor of time to tumor progression and post-progression survival in non-smokers (Figures ?(Figures9B9B and 10B), with hazard-ratios of 3.64 and 2.89, respectively.

Thus, the mitochondrial chaperone, HSPD1, is an effective predictive biomarker of overall survival and tumor progression, in both smokers and non-smokers as well.

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DISCUSSION
Linking CSC propagation with telomerase activity and mitochondrial function: Targeting CSCs with doxycycline and/or palbociclibRecently, we determined the functional role of telomerase activity in lung cancer stem cell (CSC) propagation. More specifically, we indirectly monitored telomerase activity, by linking the hTERT-promoter to eGFP [ 4, 5]. Using A549 lung cancer cells, stably-transfected with the hTERT-GFP reporter, we then used GFP-expression fluorescence intensity to fractionate these cell lines into GFP-high and GFP-low cell populations. We functionally compared the phenotype of these GFP-high and GFP-low cell sub-populations. Importantly, we directly demonstrated that cancer cells with higher telomerase activity (GFP-high) are energetically-activated, with increased mitochondrial function and increased glycolysis. This was directly confirmed by proteomics analysis. Cells with high telomerase activity showed increased stem cell activity (measured via 3D-spheroid formation) and an increased capacity for cell migration (measured with a Boyden-chamber). These phenotypes were blocked by inhibitors of energy-metabolism, which targeted either mitochondrial OXPHOS or glycolysis, or by using doxycycline, an FDA-approved antibiotic, that inhibits mitochondrial biogenesis as an off-target effect [ 4, 5].

The levels of telomerase activity also determined the ability of hTERT-high CSCs to proliferate, as assessed by measuring DNA synthesis [ 4, 5]. Treatment with Palbociclib, an FDA-approved CDK4/6 inhibitor specifically blocked the propagation of lung CSCs, at concentrations in the nanomolar range. Therefore, telomerase-high CSCs are among the most energetically activated, migratory and proliferative cell sub-populations. These observations may provide a mechanistic explanation for why long telomere length [ 69] (a surrogate marker of increased telomerase activity) is specifically associated with metastasis and poor clinical outcome in NSC lung cancer and many other tumor types. Thus, high telomerase activity may drive poor clinical outcome by activating mitochondrial biogenesis, “fueling” the proliferation in lung CSCs [ 4, 5].

Using mitochondrial markers as companion diagnostics in NSCLC patients: Importance for treatment stratification and personalized medicineConsistent with this novel hypothesis linking high telomerase activity with enhanced mitochondrial function, we show here that mitochondrial markers effectively predict poor overall survival in lung cancer patients, with negative surgical margins. Importantly, these mitochondrial markers could now be used to identify high-risk lung cancer patients at diagnosis, up to 10 years in advance. These results also suggest that mitochondria should be therapeutically-targeted in epithelial lung cancer cells to significantly extend patient survival.

In this workflow, high-risk patients should be first identified at diagnosis by the high expression of mitochondrial markers in their primary lung tumors (Figure ?(Figure11).11). Then, these patients could be treated with FDA-approved therapeutics (e.g., Doxycycline or Palbociclib; in combination with the standard of care), to improve poor overall survival. Importantly, both of these drugs have already been shown to be effective against the propagation of the lung CSC sub-population.





Figure 11
NSC lung cancer: mitochondrial-based diagnostics for personalized cancer therapyIn this diagram, mitochondrial-based diagnostics would be used to separate lung cancer patients into high-risk and low-risk groups. Then, patients with high levels of mitochondrial markers in their primary tumor (“bad prognosis”) would be treated with mitochondrial-based therapies (such as “Doxycycline”), as an add-on to the standard of care, to prevent tumor progression and increase overall survival.



In this context, these mitochondrial markers could also be used as effective companion diagnostics for new experimental therapeutics targeting either mitochondria or telomerase (hTERT) and/or cell proliferation, to select the high-risk lung cancer patient sub-group, allowing proper treatment stratification.

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MATERIALS AND METHODS
Kaplan-Meier (K-M) analysesTo perform K-M analysis on nuclear mitochondrial gene transcripts, we used an open-access online survival analysis tool to interrogate publically available microarray data from up to 1,926 lung cancer patients [ 3]. This allowed us to determine their overall prognostic value. For this purpose, we primarily analyzed 10-year follow-up data from non-small cell lung cancer (NSCLC) patients that had negative surgical margins (N = 726) [ 3]. Biased array data were excluded from the analysis. This allowed us to identify > 180 nuclear mitochondrial gene probes, with significant prognostic value. Hazard-ratios were calculated, at the best auto-selected cut-off, and p-values were calculated using the logrank test and plotted in R. K-M curves were also generated online using the K-M-plotter (as high-resolution TIFF files), using univariate analysis:

kmplot.com = service&cancer = lung.

This allowed us to directly perform in silico validation of these mitochondrial biomarker candidates. The most updated version of the database (2015) was utilized for all these analyses.

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Acknowledgments
It should be noted that this bioinformatics analysis, focused on nuclear-encoded mitochondrial-related gene transcripts, was not funded by a specific grant and did not require any research expenditures, since no “wet” laboratory experiments were performed.

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Abbreviations
CSCscancer stem-like cells
FPfirst progression
HRhazard ratio
K-MKaplan-Meier
LNlymph node
MRPLmitochondrial ribosomal proteins, large subunit
MRPSmitochondrial ribosomal proteins, small subunit
Nnumber of patients in a given data set
NSCLCnon-small cell lung cancer
OSoverall survival
OXPHOSoxidative phosphorylation (mitochondrial respiration)
PPSpost progression survival


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Footnotes

Contributed byAuthor contributions

Professor Lisanti (MPL) and Dr. Sotgia (FS) conceived and initiated this project. Professor Lisanti and Dr. Sotgia both performed the bioinformatics analysis, and wrote the manuscript.

CONFLICTS OF INTEREST

MPL and FS hold a minority interest in Lunella, Inc.

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REFERENCES

1. Yoon SM, Shaikh T, Hallman M. Therapeutic management options for stage III non-small cell lung cancer. World J Clin Oncol. 2017;8:1–20. [ PMC free article] [ PubMed]
2. Xiong Y, Huang BY, Yin JY. Pharmacogenomics of platinum-based chemotherapy in non-small cell lung cancer: focusing on DNA repair systems. Med Oncol. 2017;34:48. [ PubMed]
3. Gyorffy B, Surowiak P, Budczies J, Lanczky A. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS One. 2013;8:e82241. [ PMC free article] [ PubMed]
4. Bonuccelli G, Peiris-Pages M, Ozsvari B, Martinez-Outschoorn UE, Sotgia F, Lisanti MP. Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity. Oncotarget. 2017;8:9868–84. doi.org [ PMC free article][ PubMed]
5. Lamb R, Ozsvari B, Bonuccelli G, Smith DL, Pestell RG, Martinez-Outschoorn UE, Clarke RB, Sotgia F, Lisanti MP. Dissecting tumor metabolic heterogeneity: Telomerase and large cell size metabolically define a sub-population of stem-like, mitochondrial-rich, cancer cells. Oncotarget. 2015;6:21892–905. doi.org [ PMC free article] [ PubMed]
6. Telomeres Mendelian Randomization Collaboration. Haycock PC, Burgess S, Nounu A, Zheng J, Okoli GN, Bowden J, Wade KH, Timpson NJ, Evans DM, Willeit P, Aviv A, Gaunt TR, et al. Association Between Telomere Length and Risk of Cancer and Non-Neoplastic Diseases: A Mendelian Randomization Study. JAMA Oncol. 2017 Feb 23; doi: 10.1001/jamaoncol.2016.5945. [Epub ahead of print] [ PMC free article] [ PubMed] [ CrossRef]
7. Svenson U, Roos G, Wikström P. Long leukocyte telomere length in prostate cancer patients at diagnosis is associated with poor metastasis-free and cancer-specific survival. Tumour Biol. 2017;39:1010428317692236. [ PubMed]
8. Marión RM, López de Silanes I, Mosteiro L, Gamache B, Abad M, Guerra C, Megías D, Serrano M, Blasco MA. Common Telomere Changes during In Vivo Reprogramming and Early Stages of Tumorigenesis. Stem Cell Reports. 2017;8:460–75. [ PMC free article] [ PubMed]
9. Lv Y, Zhang Y, Li X, Ren X, Wang M, Tian S, Hou P, Shi B, Yang Q. Long telomere length predicts poor clinical outcome in esophageal cancer patients. Pathol Res Pract. 2017;213:113–18. [ PubMed]

Articles from Oncotarget are provided here courtesy of Impact Journals, LLC

ncbi.nlm.nih.gov

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To: donpat who wrote (1191)2/18/2019 3:52:24 PM
From: Savant
of 1214
 
evidence suggests eating too much, and not moving enough...will make u fat

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From: donpat2/24/2019 1:43:57 PM
of 1214
 
Cancer stem cells (CSCs): metabolic strategies for their identification and eradication

Conclusions

CSC have been regarded as the cells of origin of cancer and are crucially involved in metastatic dissemination, radioresistance and chemoresistance, and disease recurrence. Mounting experimental evidence and clinical studies indicate that metabolism is not a mere player in the tumor bioenergy machinery, but it actually orchestrates stemness by enabling cell reprogramming in response to a large repertoire of environmental conditions within the stem niche. Recently, targeting the peculiar metabolic features of CSCs has hold promise to prevent disease progression and recurrence and efficiently eradicate cancer. High-throughput data combined with large-scale drug screening represent the state of the art for the characterization of the metabolic peculiarity of CSCs and the identification of selective pharmacological targets. In this scenario, the repurposing of FDA-approved drugs represents a concrete and inexpensive opportunity to extend the pharmacological and biological properties of existing compounds, and in the meanwhile gain a better understanding of CSC action in cancer. Nevertheless, a deeper focus on the metabolic plasticity of CSCs and their ability to switch to different metabolic pathways in response to certain environmental stressors like hypoxia or chemotherapics would provide a better strategic platform to hit this biochemical malleability. Furthermore, the evaluation of the metabolic fuels, intermediates, and pathways involved in maintaining the stemness traits and implicated in CSC survival in harsh conditions could unveil novel metabolic Achille's heels to be used in a therapeutic setting. A drug-controlled process aimed at forcing CSCs to adopt a certain metabolic profile could be an effective approach to prevent the metabolic adaptability of CSCs. Indeed, targeting this drug-induced metabolic inflexibility would definitely compromise CSC survival.

Biochemical Journal (2018) 475 1611–1634 doi.org

ncbi.nlm.nih.gov

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From: donpat2/26/2019 9:58:17 AM
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NanoViricides Reaches an Agreement on Terms of License for Shingles Virus Drug Development

Published: Feb 26, 2019 7:00 a.m. ET


The Company has substantially de-risked this program towards clinical success. We have already performed successful safety assessments in a non-GLP preliminary safety/toxicology study against two precursor candidates that led to NV-HHV-101 in a standard rat animal model. In these studies, the candidates were found to be extremely safe, with no evidence of dermal topical or systemic adverse events.

marketwatch.com

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From: donpat2/27/2019 12:29:40 PM
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NanoViricides Announces $2.5 Million Financing in a Registered Direct Offering

Published: Feb 27, 2019 9:48 a.m. ET



SHELTON, Conn., Feb. 27, 2019 /PRNewswire/ -- NanoViricides, Inc. (nyse mkt:NNVC) (the "Company") a global leader in the development of highly effective antiviral therapies based on a novel nanomedicines platform (the "Company"), announced today that it has entered into a securities purchase agreement with certain institutional investors providing for the purchase and sale of 6,944,446 units at a price of $0.36 per unit. Each unit comprises 1 share of common stock and 1 five-year warrant exercisable at $0.61.

Chardan acted as the placement agent in connection with the financing. The closing of the sale of the securities is expected to take place on or about February 27, 2019, subject to the satisfaction of customary closing conditions.

The shares of common stock and warrants were offered pursuant to a shelf registration statement on Form S-3 (File No. 333-216345), which was declared effective by the United States Securities and Exchange Commission ("SEC") on April 25, 2017.

This press release does not constitute an offer to sell or the solicitation of an offer to buy, nor will there be any sales of these securities in any jurisdiction in which such offer, solicitation or sale would be unlawful prior to registration or qualification under the securities laws of such jurisdiction. A prospectus supplement relating to the shares of common stock will be filed by the Company with the SEC. When available, copies of the prospectus supplement, together with the accompanying prospectus, can be obtained at the SEC's website at www.sec.gov or from Chardan Capital, LLC, 17 State Street, NY, NY 10004, New York, New York 10022, by calling 646-465-9028.

The Company believes this financing provides valuable capital that will immediately help the Company advance its drug pipeline into human clinical trials.

Recently, on February 4, 2019, the Company has announced that it has selected a clinical candidate for further development under the HerpeCide™ broad-spectrum drug program. The candidate, called "NV-HHV-101" is now in IND-enabling cGLP Safety/Toxicology studies in the regulatory pathway. Its first indication is for the treatment of shingles rash as a dermal topical cream. In addition, our drug candidates against HSV-1 "cold sores" and HSV-2 "genital herpes" are in advanced studies and are expected to follow the shingles drug candidate into human clinical trials. Shingles in adults and chicken pox in children is caused by the same virus, namely VZV (Varicella-zoster virus, aka HHV-3 or human herpesvirus-3). Topical application has the advantage of being able to deliver very high drug concentrations locally to completely eradicate the virus. In contrast, the local concentrations and therefore effectiveness of orally delivered medications is limited by the toxicity and bioavailability of the oral drug, as is known for the existing antiviral therapies for HSV-1, HSV-2, and VZV.

The overall market addressed by the HerpeCide program is in tens of billions of dollars. in particular, there is currently no effective treatment for shingles. The market size for an effective anti-shingles drug is currently estimated to be in the range of billions of dollars, even after a new shingles vaccine, Shingrix® (GlaxoSmithKline) has been approved.

The Company has substantially de-risked this program towards clinical success. We have already performed successful safety assessments in a non-GLP preliminary safety/toxicology study against two precursor candidates that led to NV-HHV-101 in a standard rat animal model. In these studies, the candidates were found to be extremely safe, with no evidence of dermal topical or systemic adverse events.

We scaled up production after achieving these strong safety signals, to get ready for the IND-enabling cGLP Safety/Toxicology studies and the Phase I human clinical trials, at our own facility. This enabled rapid translation to regulatory development, minimized cost outlays, saved time, and improved quality assurance, as compared to going to an external contract manufacturing organization ("CMO"). Also, this strategy has de-risked the manufacture by keeping it in the hands of experts who developed the scalable chemistries and performed the scale-up operations.

The Company develops its class of drugs, that we call nanoviricides®, using a platform technology. This approach enables rapid development of new drugs against a number of different viruses. A nanoviricide is a "biomimetic" - it is designed to "look like" the cell surface to the virus. The nanoviricide® technology enables direct attacks at multiple points on a virus particle. It is believed that such attacks would lead to the virus particle becoming ineffective at infecting cells. Antibodies in contrast attack a virus particle at only a maximum of two attachment points per antibody. In addition, the nanoviricide technology also simultaneously enables attacking the rapid intracellular reproduction of the virus by incorporating one or more active pharmaceutical ingredients (APIs) within the core of the nanoviricide. The nanoviricide technology is the only technology in the world, to the best of our knowledge, that is capable of both (a) attacking extracellular virus, thereby breaking the reinfection cycle, and simultaneously (b) disrupting intracellular production of the virus, thereby enabling complete control of a virus infection.

About NanoViricides
NanoViricides, Inc. ( www.nanoviricides.com) is a development stage company that is creating special purpose nanomaterials for antiviral therapy. The Company's novel nanoviricide® class of drug candidates are designed to specifically attack enveloped virus particles and to dismantle them. The Company is developing drugs against a number of viral diseases including oral and genital Herpes, viral diseases of the eye including EKC and herpes keratitis, H1N1 swine flu, H5N1 bird flu, seasonal Influenza, HIV, Hepatitis C, Rabies, Dengue fever, and Ebola virus, among others. This press release contains forward-looking statements that reflect the Company's current expectation regarding future events. Actual events could differ materially and substantially from those projected herein and depend on a number of factors. Certain statements in this release, and other written or oral statements made by NanoViricides, Inc. are "forward-looking statements" within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934. You should not place undue reliance on forward-looking statements since they involve known and unknown risks, uncertainties and other factors which are, in some cases, beyond the Company's control and which could, and likely will, materially affect actual results, levels of activity, performance or achievements. The Company assumes no obligation to publicly update or revise these forward-looking statements for any reason, or to update the reasons actual results could differ materially from those anticipated in these forward-looking statements, even if new information becomes available in the future. Important factors that could cause actual results to differ materially from the company's expectations include, but are not limited to, those factors that are disclosed under the heading "Risk Factors" and elsewhere in documents filed by the company from time to time with the United States Securities and Exchange Commission and other regulatory authorities. Although it is not possible to predict or identify all such factors, they may include the following: demonstration and proof of principle in preclinical trials that a nanoviricide is safe and effective; successful development of our product candidates; our ability to seek and obtain regulatory approvals, including with respect to the indications we are seeking; the successful commercialization of our product candidates; and market acceptance of our products.

View original content to download multimedia:http://www.prnewswire.com/news-releases/nanoviricides-announces-2-5-million-financing-in-a-registered-direct-offering-300803271.html

SOURCE NanoViricides, Inc.

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From: donpat2/28/2019 5:42:23 PM
of 1214
 
NanoViricides (NNVC) Stock: Rocketing On Licensing Agreement

By
Joshua Rodriguez
-

February 26, 2019



NanoViricides Inc (NYSEAMERICAN: NNVC) is flying early on in the trading session this morning, and for good reason. The company announced that it has entered into a licensing agreement, exciting investors and sending the stock screaming for the top. Today, we’ll talk about:

The licensing agreement;what we’re seeing from NNVC stock as a result; andwhat we’ll be watching for ahead. NNVC Stock Climbs On Licensing AgreementAs mentioned above, NanoViricides is having a great day in the market today after announcing a licensing agreement. The agreement was announced via press release early this morning.

In the release, NNVC said that it had reached an agreement to on the terms of a license for the drug development against VZV. VZV is a virus that cuases chickenpox in children and shingles in adults.

According to the release, the company and TheraCour have agreed that TheraCour will not receive any payment upon signing of the agreement. Also, the agreement states that TheraCour will receive 500,000 shares of the company’s Series A Preferred Stock as a milestone payment when an IND becomes effective.

If the company completes Phase 1 human trials, a payment of $1.5 million will be made in cash. Upon the completion of Phase 2 human trials, a payment of $2.5 million will be made in cash. Other payments will be consistent with the company’s existing license agreements with TheraCour, which include a 15% royalty on sales and 15% of any sub-licensing revenues.

This is overwhelmingly positive news. First and foremost, it means that the company will be pushing a new asset through the development process. Moreover, NNVC got a great deal on the agreement, which won’t require cash milestone payments until the completion of Phase 1 trials.

What We’re Seeing from The Stock
One of the first lessons that we learn when we start to work in the market is that the news leads to moves. When it comes to NanoViricides, the news proved to be overwhelmingly positive.

After all, the new agreement will open the doors to the development of a new product, one that is much needed in the field of medicine. So, it’s not surprising to see that excited investors are pushing the stock up in the market this morning.

As is normally the case, our partners at Trade Ideas were the first to alert us to the gains. Currently (9:11), NNVC is trading at $0.71 per share after a gain of $0.29 per share or 70.26% thus far today.

Stop wasting your time! Start finding winning trades in minutes with Trade Ideas!

What We’ll Be Watching For Ahead
Moving forward, the CNA Finance team will continue to keep a close eye on NNVC. In particular, we’re interested in following the story surrounding the company’s work to expand the development of its pipeline. Nonetheless, we’ll keep a close eye on the news and bring it to you as it breaks!

cnafinance.com

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From: donpat3/3/2019 9:57:28 AM
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From: donpat3/5/2019 8:27:15 AM
of 1214
 
NanoViricides Reports Excellent Safety Profile of Its First Drug Candidate in Dermal Treatment Study

Published: Mar 5, 2019 7:00 a.m. ET

SHELTON, Conn., Mar. 5, 2019 /PRNewswire/ -- NanoViricides, Inc. (nyse mkt:NNVC) (the "Company") a company with novel platform technology to treat difficult and life-threatening viral diseases, announces that its first drug candidate has successfully completed the first of the Safety/Toxicology studies moving towards human clinical trials.

The Company is developing its broad-spectrum drug candidate in the HerpeCide™ program, namely, "NV-HHV-101," towards human clinical trials. The drug candidate is formulated as a dermal topical cream.The first part of the IND-enabling safety/toxicology studies comprised a non-GLP evaluation of safety and tolerability of the drug candidate in mini-pigs. The study was performed by BASi, Evansville, IN, an independent contract research organization that is specialized in IND-enabling safety/toxicology studies.

In this study, all dosage levels of the nanoviricide® drug product, including the maximum feasible dose, were well tolerated in all treated animals. All of the parameters evaluated in the study remained within normal ranges, and showed no adverse effects from the drug treatment.

These strong safety results are consistent with our previous studies on two related drug candidates during drug candidate optimization. The Company has previously tested two related development candidates in the HerpeCide™ program in non-GLP Safety and Tolerability study in rats for dermal exposure as well as systemic exposure by subcutaneous and intravenous routes of administration. The Company previously reported that those candidates were both found to be safe based on multiple parameters in the completed non-GLP Tolerability study.

Mini-pigs are considered to be a well correlated animal model for human dermal drug and cosmetics development. Pigs and mini-pigs are a much closer correlate of human clinical results as compared to rats for dermal topical applications.

The success of these studies forms the basis for progressing the drug candidate into more formal GLP Safety/Toxicology studies. The Company is negotiating with BASi for the earliest possible date for starting the GLP phase of these IND-enabling studies. Assuming that the GLP studies are also successful, the Company anticipates advancing NV-HHV-101 into human clinical trials for topical dermal treatment of the shingles rash as the initial indication.

In this non-GLP study, the drug skin cream was applied topically twice daily to approximately 10% of the total body surface area of each mini-pig in the group for seven days, as per a standard protocol for regulatory agencies including the US FDA. Each group of animals received a single dosage level (i.e. a given concentration of the API in the same amount of formulation vehicle). The dosage levels were varied from zero (vehicle alone) up to the maximum feasible dose in the study employing multiple groups.

The animals were evaluated daily for general signs of adverse effects and toxicity including body weight, detailed clinical physical observations as well as the specific evaluation of the skin treatment areas. Prior to treatment and one day after completion of dosing, blood samples were taken from each animal for hematology and clinical chemistry standard testing to assess systemic adverse effects, if any.

The seven days of treatment in this study began towards the end of December 2018, as previously reported by the Company.

Minipig size, anatomy, physiology, metabolism, and genetics are considered to correlate well to those of humans. Pig skin is anatomically, physiologically, biochemically, and immunologically similar to human skin. Also, pig skin is fixed or adherent like humans unlike the loose skin of rodents and dogs that are typically used in other safety and tolerability studies. The outer skin layer, the epidermis, of the minipig is also approximately the same thickness as that of human skin. Thus, minipigs are preferred by regulatory agencies for dermal treatment safety and tolerability studies.

The Company also continues to evaluate this broad-spectrum drug candidate as well as certain variations based on the same candidate, for the treatment of other herpesviruses, namely HSV-1 cold sores and HSV-2 genital herpes. The market size for our immediate target drugs in the HerpeCide™ program is variously estimated into billions to tens of billions of dollars. The Company believes that its dermal topical cream for the treatment of shingles rash will be its first drug heading into clinical trials. The Company believes that additional topical treatment candidates in the HerpeCide™ program, namely, HSV-1 "cold sores" treatment, and HSV-2 "genital ulcers" treatment are expected to follow the shingles candidate into IND-enabling development and then into human clinical trials.

About NanoViricides
NanoViricides, Inc. ( www.nanoviricides.com) is a development stage company that is creating special purpose nanomaterials for antiviral therapy. The Company's novel nanoviricide® class of drug candidates are designed to specifically attack enveloped virus particles and to dismantle them. The Company is developing drugs against a number of viral diseases including H1N1 swine flu, H5N1 bird flu, seasonal Influenza, HIV, oral and genital Herpes, viral diseases of the eye including EKC and herpes keratitis, Hepatitis C, Rabies, Dengue fever, and Ebola virus, among others. This press release contains forward-looking statements that reflect the Company's current expectation regarding future events. Actual events could differ materially and substantially from those projected herein and depend on a number of factors. Certain statements in this release, and other written or oral statements made by NanoViricides, Inc. are "forward-looking statements" within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934. You should not place undue reliance on forward-looking statements since they involve known and unknown risks, uncertainties and other factors which are, in some cases, beyond the Company's control and which could, and likely will, materially affect actual results, levels of activity, performance or achievements. The Company assumes no obligation to publicly update or revise these forward-looking statements for any reason, or to update the reasons actual results could differ materially from those anticipated in these forward-looking statements, even if new information becomes available in the future. Important factors that could cause actual results to differ materially from the company's expectations include, but are not limited to, those factors that are disclosed under the heading "Risk Factors" and elsewhere in documents filed by the company from time to time with the United States Securities and Exchange Commission and other regulatory authorities. Although it is not possible to predict or identify all such factors, they may include the following: demonstration and proof of principle in preclinical trials that a nanoviricide is safe and effective; successful development of our product candidates; our ability to seek and obtain regulatory approvals, including with respect to the indications we are seeking; the successful commercialization of our product candidates; and market acceptance of our products.

FDA refers to US Food and Drug Administration. "API" means active pharmaceutical ingredient.

View original content to download multimedia:
prnewswire.com

SOURCE NanoViricides, Inc.

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From: donpat3/5/2019 8:33:16 AM
of 1214
 
NanoViricides announces $2.5M financing in registered direct offering

MICHELLE SULLO

FEBRUARY 27, 2019

NanoViricides Inc., a Shelton-based company that is developing nanotechnology based antiviral medicines, announced Wednesday it is entering into a securities purchase agreement with institutional investors.

The agreement provides for the purchase and sale of over 6.9 million units at a price of $0.36 per unit, according to a press release. Each unit represents one share of common stock and one five-year warrant exercisable at $0.61.

The company is developing medication aimed at viral diseases such as influenza, swine flu, bird flu, HIV/AIDS, cold sores and genital herpes, viral eye diseases, dengue viruses, Ebola virus, rabies, and more.

In early February, the company announced it had selected a clinical candidate for further development under the HerpeCide broad-spectrum drug program, a topical skin cream expected to be effective for treating the shingles rash. Shingles in adults is caused by the same virus as the chicken pox in children, and there currently is no effective treatment for it.

"The market size for an effective anti-shingles drug is currently estimated to be in the range of billions of dollars, even after a new shingles vaccine, Shingrix (GlaxoSmithKline) has been approved," the company said in a press release.

According to company officials, their drug candidates against cold sores and genital herpes are in advanced studies and are expected to follow the shingles drug candidate into human clinical trials.

The investment bank Chardan acted as the placement agent in connection with the financing, according to NanoViricides, which expected the closing of the sale of the securities to take place on Wednesday.

NanoViricides officials believe this financing will provide key capital to immediately help it advance its drugs into human clinical trials.

The company will file a prospectus supplement related to the shares of common stock with the U.S. Securities and Exchange Commission, which will be available at www.sec.gov.

m.hartfordbusiness.com

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